Method for the production of L-serine using genetically engineered microorganisms deficient in serine degradation pathways

ABSTRACT

The present invention generally relates to the microbiological industry, and specifically to the production of L-serine using genetically modified bacteria. The present invention provides genetically modified microorganisms, such as bacteria, wherein the expression of genes encoding for enzymes involved in the degradation of L-serine is attenuated, such as by inactivation, which makes them particularly suitable for the production of L-serine at higher yield. The present invention also provides means by which the microorganism, and more particularly a bacterium, can be made tolerant towards higher concentrations of serine. The present invention also provides methods for the production of L-serine or L-serine derivative using such genetically modified microorganisms.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase Application of PCTInternational Application Number PCT/EP2016/051701, filed on Jan. 27,2016, designating the United States of America and published in theEnglish language, which is an International Application of and claimsthe benefit of priority to European Patent Application No. 15152643.1,filed on Jan. 27, 2015. The disclosures of the above-referencedapplications are hereby expressly incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing isSeqList-ZACCO44-016APC.txt, the date of creation of the ASCII text fileis Feb. 2, 2016, and the size of the ASCII text file is 129 KB.

TECHNICAL FIELD OF THE INVENTION

The present invention generally relates to the microbiological industry,and specifically to the production of L-serine using geneticallymodified bacteria. The present invention provides genetically modifiedmicroorganisms, such as bacteria, wherein the expression of genesencoding for enzymes involved in the degradation of L-serine isattenuated, such as by inactivation, which makes them particularlysuitable for the production of L-serine at higher yield. The presentinvention also provides means by which the microorganism, and moreparticularly a bacterium, can be made tolerant towards higherconcentrations of serine. The present invention also provides methodsfor the production of L-serine or L-serine derivative using suchgenetically modified microorganisms.

BACKGROUND OF THE INVENTION

L-serine is an amino acid that currently is used in the cosmetics,pharmaceutical and medical industry. The estimated annual production ofserine is between 300-1000 tons (Leuchtenberger et al., 2005). Thecompound has also been identified as one of the top 30 most interestingbiochemicals because of its potential use as a building blockbiochemical. The current production is based on conversion of glycineand methanol using resting cells (Hagishita et al., 1996), wheremethylotrophs convert methanol to fomaldehyde and transfer the CH₂—OHunit of the molecule to glycine using serine hydroxymethyltransferase(glyA). This fermentation process is time consuming and glycine is anexpensive starting material. Developing a method for producing serine atlow cost directly from glucose is therefore attractive.

Serine has the potential to be made from glucose by fermentation with avery high theoretical yield (Burgard and Maranas, 2001). However,several challenges need to be addressed in order to increase the yield,the most crucial one being degradation of serine in the productionorganism.

Serine has two key degradation pathways in E. coli. Serine to pyruvatecatabolism is in E. coli catalyzed by three deaminases namely sdaA, sdaBand tdcG, while C. glutamicum only has one deaminase (sdaA) withactivity towards serine. In both organisms, the conversion of serine toglycine is encoded by glyA. Serine production by knocking out onlydeaminases has been attempted in E. coli (U et al., 2012) and C.glutamicum (Peters-Wendisch et al., 2005). In E. coli transientaccumulation of 3.8 mg/L from 1 g/L glucose was observed when only oneof the pathway gene (serA) was overexpressed. Deletion of the deaminaseon C. glutamicum lead to marginal and transient increase in the serinetiter. In recent studies, E. coli was engineered to enhance the flux of3-phosphoglycerate by perturbing the TCA-cycle and glyoxylate shunt (Guet al., 2014). The resulting strain, where only one deaminase, wasremoved (sdaA) was reported to produce 8.45 g/L serine from 75 g/Lglucose (11.2% yield).

Down regulation of glyA (Peters-Wendisch et al., 2005) in C. glutamicumresulted in the production of 9 g/L serine from 40 g/L glucose but leadto an unstable strain. glyA is an important enzyme that converts serineto glycine and in this step transfers one carbon unit to tetrahydrofolate (THF), which is used as cofactor. Removal of the folic acidpathway and supplementation of folic acid lead to a stable C.glutamicum, and a production of 36 g/L serine, however with a relativelylow yield (Stolz et al, 2007).

Deletion of both of the major serine degradation pathways (serine topyruvate and serine to glycine) has not been previously been achieved.It is furthermore known that serine becomes toxic even at lowconcentrations in strains that lack the pyruvate degradation pathway(Zhang and Newman, 2008). It is expected that serine may inhibit theproduction of branched amino acids in E. coli Hama et al., 1990), andthe conversion of serine to hydroxypyruvate and acrylates, which istoxic to the cell (de Lorenzo, 2014). For efficient production ofL-serine or a derivative thereof, it is therefore desirable to bothremove the serine degradation pathways and address the problemsassociated with toxicity of serine.

SUMMARY OF THE INVENTION

The object of the present invention is to provide means allowing a moreefficient production of L-serine. More particularly, it is an object ofthe present invention to provide means allowing the production ofL-serine at higher nominal yield and improved mass yield.

This is achieved by the finding that the production of L-serine can beenhanced by, e.g., inactivation of genes encoding enzymes involved inthe degradation of L-serine, notably the genes sdaA, sdaB, tdcG andglyA.

The present invention thus provides in a first aspect a bacterium,especially a bacterium having an ability to produce L-serine, whereinsaid bacterium has been modified to attenuate expression of genes codingfor polypeptides having serine deaminase activity and to attenuateexpression of a gene coding for a polypeptide having serinehydroxymethyltransferase activity.

More particularly, the present invention provides a bacterium which hasbeen modified to attenuate the expression of the genes sdaA, sdaB, tdcGand glyA, e.g., by inactivation of these genes.

The present invention provides in a second aspect a method for producingL-serine comprising: cultivating the bacterium as described above in amedium.

The present invention provides in a further aspect a yeast, such asSaccharomyces cerevisiae, especially a yeast having an ability toproduce L-serine, wherein said yeast has been modified to attenuateexpression of a gene coding for a polypeptide having serine deaminaseactivity and to attenuate expression of genes coding for polypeptideshaving serine hydroxymethyltransferase activity.

More particularly, the present invention provides a yeast, Saccharomycescerevisiae, which has been modified to attenuate the expression of thegenes CHA1, SHM1 and SHM2, e.g., by inactivation of these genes.

The present invention provides in a further aspect a method forproducing L-serine or a L-serine derivative comprising:

cultivating the yeast as described above in a culture medium.

The present invention provides in a further aspect a (isolated) nucleicacid molecule, such as a vector, comprising a nucleotide sequenceencoding a polypeptide having an amino acid sequence which has at leastabout 90%, such as at least about 93%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%, sequence identity to the amino acid sequence set forth in SEQ IDNO: 11 which comprises an amino acid substitution at position Y356, S357and/or S359.

The present invention provides in a further aspect a (isolated)polypeptide having an amino acid sequence which has at least about 90%,such as at least about 93%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position Y356, S357 and/or S359.The (isolated) polypeptide may be one expressed by (and isolated from) abacterium of the invention.

The present invention can be summarized by the following items:

1. A bacterium which has been modified to attenuate expression of genescoding for polypeptides having serine deaminase activity and toattenuate expression of a gene coding for a polypeptide having serinehydroxymethyltransferase activity.

2. The bacterium according to item 1, wherein the expression of thegenes sdaA, sdaB, tdcG and glyA is attenuated.

3. The bacterium according to item 1 or 2, wherein the expression of thegenes is attenuated by inactivation of the genes.

4. The bacterium according to any one of items 1 to 3, wherein saidbacterium has been further modified to overexpress a 3-phosphoglyceratedehydrogenase, a phosphoserine phosphatase and a phosphoserineaminotransferase.

5. The bacterium according to any one of claims 1 to 4, wherein saidbacterium comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding a 3-phosphoglycerate dehydrogenase.

6. The bacterium according to any one of items 1 to 5, wherein saidbacterium comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding a 3-phosphoserine aminotransferase.

7. The bacterium according to any one of items 1 to 6, wherein saidbacterium comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding a phosphoserine phosphatase.

8. The bacterium according to any one of items 5 to 7, wherein theexogenous nucleic acid molecule(s) is an expression vector.

9. The bacterium according to any one of items 5 to 7, wherein theexogenous nucleic acid is stably integrated into the genome of thebacterium.

10. The bacterium according to any one of items 1 to 9, wherein saidbacterium is capable of growing in a minimal culture medium comprisingL-serine at a concentration of at least about 6.25 g/L.

11. The bacterium according to any one of items 1 to 10, wherein saidbacterium is capable of growing in a minimal culture medium comprisingL-serine at a concentration of at least about 6.25 g/L at a growth rateof at least 0.1 hr⁻¹ during exponential growth.

12. The bacterium according to any one of items 1 to 11, wherein saidbacterium has been further modified to overexpress the gene ydeD.

13. The bacterium according to any one of items 1 to 12, wherein saidbacterium comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding the protein product of the gene ydeD.

14. The bacterium according to item 13, wherein the exogenous nucleicacid molecule is an expression vector.

15. The bacterium according to item 13, wherein the exogenous nucleicacid is stably integrated into the genome of the bacterium.

16. The bacterium according to any one of items 1 to 15, wherein saidbacterium comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position Y356, one or more nucleotide substitutionsresulting in an amino acid substitution in the encoded polypeptide atposition S357 and/or one or more nucleotide substitutions resulting inan amino acid substitution in the encoded polypeptide at position S359;wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N,Y3560, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R, Y356L; thesubstitution at position S357 is selected from the group consisting ofS357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

17. The bacterium according to any one of items 1 to 16, wherein saidbacterium comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position Y356, wherein the substitution at position Y356is selected from the group consisting of Y356C, Y356T, Y356V, Y356S,Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P,Y356H, Y356R, Y356L.

18. The bacterium according to any one of items 1 to 17, wherein saidbacterium comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position S357, wherein the substitution at position S357is selected from the group consisting of S357R, S357V, S357P, S357G,S357I, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M.

19. The bacterium according to any one of items 1 to 18, wherein saidbacterium comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position S359, wherein the substitution at position S359is selected from the group consisting of S359R, S359G, S359M, S359F,S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

20. The bacterium according to any one of items 1 to 19, wherein saidbacterium comprises within the thrA gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions selectedfrom the group consisting of Y356C, S357R and S359R.

21. The bacterium according to any one of items 1 to 20, wherein saidbacterium expresses a aspartate kinase I/homoserine dehydrogenase I(ThrA) mutant having one or more amino add substitutions selected fromthe group consisting of Y356C, S357R and S359R.

22. The bacterium according to any one of items 1 to 15, wherein saidbacterium comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding a polypeptide having an amino acid sequencewhich has at least about 90% sequence identity to the amino acidsequence set forth in SEQ ID NO: 11 which comprises an amino acidsubstitution at position Y356, S357 and/or S359.

23. The bacterium according to claim 22, wherein the substitution atposition Y356 is selected from the group consisting of Y356C, Y356T,Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A,Y356I, Y356P, Y356H, Y356R and Y356L; the substitution at position S357is selected from the group consisting of S357R, S357V, S357P, S357G,S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; andthe substitution at position S359 is selected from the group consistingof S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C,S359K, S359E and S359L

24. The bacterium according to any one of items 1 to 23, wherein saidbacterium comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding a polypeptide having an amino acid sequencewhich has at least about 90% sequence identity to the amino acidsequence set forth in SEQ ID NO: 11 which comprises an amino acidsubstitution at position Y356.

25. The bacterium according to item 24, wherein the substitution atposition Y356 is selected from the group consisting of Y356C, Y356T,Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A,Y356I, Y356P, Y356H, Y356R, Y356L.

26. The bacterium according to any one of items 1 to 25, wherein saidbacterium comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding a polypeptide having an amino acid sequencewhich has at least about 90% sequence identity to the amino acidsequence set forth in SEQ ID NO: 11 which comprises an amino acidsubstitution at position S357.

27. The bacterium according to item 26, wherein the substitution atposition S357 is selected from the group consisting of S357R, S357V,S357P, S357G, S357I, S357Y, S357A, S357N, S357F, S357H, S357K, S357I andS357M.

28. The bacterium according to any one of items 1 to 25, wherein saidbacterium comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding a polypeptide having an amino acid sequencewhich has at least about 90% sequence identity to the amino acidsequence set forth in SEQ ID NO: 11 which comprises an amino acidsubstitution at position S359.

29. The bacterium according to item 28, wherein the substitution atposition S359 is selected from the group consisting of S359R, S359G,S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E andS359L

30. The bacterium according to any one of items 1 to 29, wherein saidbacterium comprises within the Irp gene one or more nucleotidesubstitutions resulting in an amino acid substitution, such asnon-conservative amino acid substitution, at position D143, such as theamino acid substitution D143G, in the encoded polypeptide.

31. The bacterium according to any one of items 1 to 30, wherein saidbacterium comprises within the rho gene one or more nucleotidesubstitutions resulting in an amino acid substitution, such asnon-conservative amino acid substitution, at position R87, such as theamino acid substitution R87L, in the encoded polypeptide.

32. The bacterium according to any one of items 1 to 31, wherein saidbacterium comprises within the eno gene one or more nucleotidesubstitutions resulting in an amino acid substitution, such asnon-conservative amino acid substitution, at position V164, such as theamino acid substitution V164L, in the encoded polypeptide.

33. The bacterium according to any one of items 1 to 32, wherein saidbacterium comprises within the argP gene one or more nucleotidesubstitutions resulting in an amino acid substitution, such asnon-conservative amino acid substitution, at position Q132, such as theamino acid substitution Q132K, in the encoded polypeptide.

34. The bacterium according to any one of items 1 to 33, wherein saidbacterium comprises within the tufA gene one or more nucleotidesubstitutions resulting in an amino acid substitution, such asnon-conservative amino acid substitution, at position G19, such as theamino acid substitution G19V, in the encoded polypeptide.

35. The bacterium according to any one of items 1 to 34, wherein saidbacterium comprises within the cycA gene one or more nucleotidesubstitutions resulting in an amino acid substitution, such asnon-conservative amino acid substitution, at position I220, such as theamino acid substitution 1220V, in the encoded polypeptide.

36. The bacterium according to any one of items 1 to 35, wherein saidbacterium comprises within the rpe gene one or more nucleotidesubstitutions resulting in an amino acid substitution, such asnon-conservative amino acid substitution, at position I202, such as theamino acid substitution I202T, in the encoded polypeptide.

37. The bacterium according to any one of items 1 to 36, wherein saidbacterium comprises within the yojl gene one or more nucleotidesubstitutions resulting in an amino acid substitution, such asnon-conservative amino acid substitution, at position D334, such as theamino acid substitution D334H, in the encoded polypeptide.

38. The bacterium according to any one of Items 1 to 37, wherein saidbacterium comprises within the hyaF gene one or more nucleotidesubstitutions resulting in an amino acid substitution, such asnon-conservative amino acid substitution, at position V120, such as theamino acid substitution V120G, in the encoded polypeptide.

39. The bacterium according to any one of items 1 to 38, wherein saidbacterium has been further modified to attenuate expression of the genepykF (e.g., by inactivation of the gene).

40. The bacterium according to any one of items 1 to 39, wherein saidbacterium has been further modified to attenuate expression of the genemalT (e.g., by inactivation of the gene).

41. The bacterium according to any one of items 1 to 40, wherein saidbacterium comprises within the rpoB gene one or more nucleotidesubstitutions resulting in an amino acid substitution, such asnon-conservative amino acid substitution, at position P520, such as theamino acid substitution P5201L, in the encoded polypeptide.

42. The bacterium according to any one of items 1 to 41, wherein saidbacterium comprises within the fumB gene one or more nucleotidesubstitutions resulting in an amino acid substitution, such asnon-conservative amino acid substitution, at position T218, such as theamino add substitution T218P, in the encoded polypeptide.

43. The bacterium according to any one of items 1 to 42, wherein saidbacterium comprises within the gshA gene one or more nucleotidesubstitutions resulting in an amino acid substitution, such asnon-conservative amino acid substitution, at position A178, such as theamino acid substitution A178V, in the encoded polypeptide.

44. The bacterium according to any one of items 1 to 43, wherein saidbacterium has been further modified to attenuate expression of the genelamB (e.g., by inactivation of the gene).

45. The bacterium according to any one of items 1 to 44, wherein saidbacterium comprises within its genome a deletion of about 2854 bp from alocation which corresponds to location 850092 in the E. coli K12 MG1655reference genome deposited under NCBI accession number NC_000913.2.

46. The bacterium according to item 45, wherein the deletion results indeletion of the first 5 bp of rhtA, complete deletion of genes ompX andybiP, deletion of 239 bp of sRNA rybA and 77 bp deletion of mnt5 gene.

47. The bacterium according to any one of items 1 to 46, wherein thebacterium comprises within its genome an insertion of an 768 bp longinsertion sequence element IS1 in the lagging strand at a location whichcorresponds to location 3966174 in the E. coli K12 MG1655 referencegenome deposited under NCBI accession number NC_000913.2.

48. The bacterium according to any one of items 1 to 47, wherein thebacterium comprises within its genome an insertion of 1 bp at a locationwhich corresponds to location 2942629 in the E. coli K12 MG1655reference genome deposited under NCBI accession number NC_000913.2.

49. The bacterium according to any one of items 1 to 48, wherein thebacterium comprises within its genome an insertion of a 1342 bp longinsertion sequence element IS54 at a location which corresponds tolocation 2942878 in the E. coli K12 MG1655 reference genome depositedunder NCBI accession number NC_000913.2.

50. The bacterium according to any one of items 1 to 49, the bacteriumcomprises within its genome an insertion of 1 bp at a location whichcorresponds to location 2599854 in the E. coli K12 MG1655 referencegenome deposited under NCBI accession number NC_000913.2.

51. The bacterium according to any one of items 1 to 51, wherein thebacterium comprises within its genome an insertion of an 768 bp longinsertion sequence element IS1 in the lagging strand at a location whichcorresponds to location 2492323 in the E. coli K12 MG1655 referencegenome deposited under NCBI accession number NC_000913.2.

52. The bacterium according to any one of items 1 to 51, wherein thebacterium comprises within its genome an insertion of an 1195 bp longinsertion sequence element IS5 at a location which corresponds tolocation 121518 in the E. coli K12 MG1655 reference genome depositedunder NCBI accession number NC_000913.2.

53. The bacterium according to any one of items 1 to 52, wherein thebacterium comprises within its genome an insertion of an 768 bp longinsertion sequence element IS1 in the lagging strand at a location whichcorresponds to location 1673670 in the E. coli K12 MG1655 referencegenome deposited under NCBI accession number NC_000913.2.

54. The bacterium according to any one of items 1 to 53, wherein saidbacterium has been modified to attenuate expression of a gene coding fora polypeptide having Glucose 6-phosphate-1-dehydrogenase (G6PDH)activity.

55. The bacterium according to item 54, wherein the expression of thezwf gene is attenuated.

56. The bacterium according to item 54 or 55, wherein the expression ofthe gene is attenuated by inactivation of the gene.

57. The bacterium according to any one of items 1 to 56, wherein saidbacterium expresses a polypeptide encoded by the brnQ gene, wherein saidpolypeptide terminates after position 308 or any position upstreamthereof.

58. The bacterium according to any one of items 54 to 57, wherein saidbacterium further expresses a polypeptide encoded by the thrA gene,wherein in said polypeptide at position 357 serine is replaced byanother amino acid, such as arginine.

59. The bacterium according to any one of items 54 to 58, wherein saidbacterium expresses a polypeptide encoded by the rho gene, wherein insaid polypeptide at position 87 R is replaced by another amino acid,such as L.

60. The bacterium according to any one of items 54 to 58, wherein saidbacterium expresses a polypeptide having the amino acid sequence setforth in SEQ ID NO: 11 or a polypeptide having at least about 90%, atleast about 93%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 11, wherein in said aminoacid sequence at position 357 serine is replaced by another amino acid,such as arginine; expresses a polypeptide having the amino acid sequenceset forth in SEQ ID NO: 13 or a polypeptide having the amino acidsequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 13, wherein in said amino acid sequence at position87 R is replaced by another amino acid, such as L; and expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 32 ora polypeptide having the amino acid sequence which has at least about90%, at least about 93%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 32.

61. The bacterium according to any one of Items 1 to 56, wherein saidbacterium has been further modified to attenuate expression of the genebrnQ (e.g., by inactivation of the gene).

62. The bacterium according to item 61, wherein said bacterium expressesa polypeptide having the amino acid sequence set forth in SEQ ID NO: 11or a polypeptide having at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 11, wherein in said amino acid sequence at position357 serine is replaced by another amino acid, such as arginine.

63. The bacterium according to item 61 or 62, wherein said bacteriumexpresses a polypeptide having the amino acid sequence set forth in SEQID NO: 13 or a polypeptide having the amino acid sequence which has atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 13,wherein in said amino acid sequence at position 87 R is replaced byanother amino acid, such as L.

64. The bacterium according to any one of items 1 to 63, wherein saidbacterium belongs to the Enterobacteriaceae family.

65. The bacterium according to item 64, wherein said bacterium belongsto a genus selected from the group consisting of Escherichia,Arsenophonus, Biostraticola, Brenneria, Buchnera, Budvicia,Buttiauxella, Cedecea, Citrobacter, Cosenzaea, Cronobacter, Dickeya,Edwardsiella, Enterobacter, Erwinia, Ewingella, Gibbsiella, Hofnia,Klebsiella, Leclercia, Leminorella, Lonsdalea, Mangrovibacter,Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium,Phaseolibacter, Photorhabdus, Plesiomonas, Proteus, Rahnella,Raoultella, Sacchorobacter, Salmonella, Samsonia, Serrtia, Shimwellia,Sodalis, Tatumella, Thorsellia, Trabulsiella, Wigglesworthia, Yersiniaand Yokenella.

66. The bacterium according to item 64 or 65, wherein said bacteriumbelongs to the genus Escherichia.

67. The bacterium according to item 66, wherein said bacterium isEscherichia coll.

68. A method for producing L-serine or a L-serine derivative, the methodcomprises cultivating a bacterium according to any one of items 1 to 67in a culture medium.

69. The method according to item 68, wherein the method is for producingL-serine.

70. The method according to item 68, wherein the method is for producingan L-serine derivative.

71. The method according to item 68, wherein the L-serine derivative isselected from the group consisting of L-cysteine, L-methionine,L-glycine, O-acetylserine, L-tryptophan, thiamine, ethanolamine andethylene glycol.

72. The method according to item 68, wherein the method is for producingL-cysteine.

73. The method according to item 68, wherein the method is for producingL-methionine.

74. The method according to item 68, wherein the method is for producingL-glycine.

75. The method according to item 68, wherein the method is for producingO-acetylserine.

76. The method according to item 68, wherein the method is for producingL-tryptophan.

77. The method according to item 68, wherein the method is for producingthiamine.

78. The method according to item 68, wherein the method is for producingethanolamine.

79. The method according to item 68, wherein the method is for producingethylene glycol.

80. The method according to any one of items 68 to 80, the methodfurther comprises collecting L-serine or the L-serine derivative fromthe culture medium.

81. A yeast which has been modified to attenuate expression of a genecoding for polypeptide having serine deaminase activity and to attenuateexpression of genes coding for polypeptides having serinehydroxymethyltransferase activity.

82. The yeast according to item 81, wherein the expression of the genesCHA1, SHM1 and SHM2 is attenuated.

83. The yeast according to item 81 or 82, wherein the expression of thegenes is attenuated by inactivation of the genes.

84. The yeast according to any one of items 81 to 83, wherein said yeasthas been further modified to overexpress a 3-phosphoglyceratedehydrogenase, a phosphoserine phosphatase and a phosphoserineaminotransferase.

85. The yeast according to any one of items 81 to 84, wherein the yeastbelongs to the genus Saccharomyces.

86. The yeast according to item 85, wherein the yeast is Saccharomycescerevisiae.

87. A method for producing L-serine or an L-serine derivative,comprising:

cultivating the yeast according to any one of items 81 to 86 in aculture medium.

88. A (isolated) nucleic acid molecule, such a vector, comprising anucleotide sequence encoding a polypeptide having an amino acid sequencewhich has at least about 90%, such as at least about 93%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99%, sequence identity to the amino acid sequence set forthin SEQ ID NO: 11 which comprises an amino acid substitution at positionY356, S357 and/or S359.

89. The (isolated) nucleic acid molecule according to item 88, whereinthe polypeptide has the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position Y356, S357 and/orS359.

90. The (isolated) nucleic acid molecule according to item 88 or 89,wherein the amino acid substitution is at position Y356.

91. The (isolated) nucleic acid molecule according to any one of items88 to 90, wherein the amino acid substitution is at position S357.

92. The (isolated) nucleic acid molecule according to any one of items88 to 91, wherein the amino acid substitution is at position S359.

93. The (isolated) nucleic acid molecule according to any one of items88 to 92, wherein the substitution at position Y356 is selected from thegroup consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G,Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L;the substitution at position S357 is selected from the group consistingof S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

94. A (isolated) polypeptide having an amino acid sequence which has atleast about 90%, such as at least about 93%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity to the amino acid sequence set forth in SEQID NO: 11 which comprises an amino acid substitution at position Y356,S357 and/or S359.

95. The (isolated) polypeptide according to item 94, where thepolypeptide has the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position Y356, 5357 and/or S359.

96. The (isolated) polypeptide according to item 94 or 95, wherein theamino acid substitution is at position Y356.

97. The (isolated) polypeptide according to any one of items 94 to 96,wherein the amino acid substitution is at position S357.

98. The (isolated) polypeptide according to any one of items 94 to 97,wherein the amino acid substitution is at position S359.

99. The (isolated) polypeptide according to any one of items 94 to 98,wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N,Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; thesubstitution at position S357 is selected from the group consisting ofS357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Deletion of the main genes involved in serine degradation, sdaA,sdaB, tdcG and glyA in E. coli. The removal of the four genes wasdemonstrated using PCR with primers specific to the relevant genes.

FIG. 2: Vector maps of the constructs (Example 2)

FIG. 3: Serine production during batch fermentation in shake flasks.

FIG. 4: Increased tolerance towards serine can be achieved byoverexpression of ydeD, a potential serine transporter. The figure showsthe growth of the cells in the presence of various concentrations ofserine.

FIG. 5: Increased tolerance towards serine can be achieved by randommutagenesis. Growth rates of the parent strain (Q1) and evolved strainsare shown at different serine concentrations.

FIG. 6: Adaptive Laboratory Evolution (ALE) experiment for improvingtolerance towards serine. (A) Growth rates during the evolutionexperiment. (B) Improved growth of evolved strains in the presence ofhigh concentrations of serine.

FIG. 7: The effect of mutations in thrA on tolerance towards serine.Three specific mutations of thrA (Y356C, S357R, S359R) were introducedinto the Q1 background and the growth of the clones was compared to thegrowth of the Q1 strain in the presence of 6.25 g/L of serine.

FIG. 8: Identification of mutations that cause increased tolerancetowards serine. Amplicon sequencing analysis was used to analyze theeffect of ALE mutations after introduction into the Q1 (DE3) strain byMAGE.

FIG. 9: (A) Serine production and cell density (OD 600 nm) measured atdifferent time points during fed batch fermentation of the Q1(DE3) andQ3(DE3) strains. (B) Production of serine from the glucose fed to thefermentor. The slope of the curve indicates the mass yield during thefermentation.

FIG. 10: (A) Cell density and serine titer of ALE 8-8 (DE3) strain. (B)Mass yield from glucose.

FIG. 11: Growth rate of mutant E. coli strains having different aminoacid substitutions observed at positions 356 (11A), 357 (11B) and 359(11C) of ThrA, respectively (the amino acid substitution is denoted bythe respective one-letter code). The growth rate of E. coli carrying thewild type thrA gene is denoted “wt”.

The present invention is now described in more detail below.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific termsused have the same meaning as commonly understood by a skilled artisanin the fields of biochemistry, genetics, and microbiology.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willprevail. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, and recombinantDNA, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Current Protocolsin Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc,Library of Congress, USA); Molecular Cloning: A Laboratory Manual, ThirdEdition, (Sambrook et al, 2001, Cold Spring Harbor, New York: ColdSpring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gaited., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Harries & S. J. Higgins eds. 1984); TranscriptionAnd Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture OfAnimal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); ImmobilizedCells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelsonand M. Simon, eds.-in-chief, Academic Press, Inc., New York),specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “GeneExpression Technology” (D. Goeddel, ed.); Gene Transfer Vectors ForMammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold SpringHarbor Laboratory); Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

Bacterium of the Invention

As indicated above, the present invention is inter alia based on thefinding that the production of L-serine can be enhanced by, e.g.,inactivation of genes encoding enzymes involved in the degradation ofL-serine, notably the genes sdaA, sdaB, tdcG and glyA.

Accordingly, the present invention provides a bacterium, especially abacterium having an ability to produce L-serine, wherein said bacteriumhas been modified to attenuate expression of genes coding forpolypeptides having serine deaminase activity and to attenuateexpression of a gene coding for a polypeptide having serinehydroxymethyltransferase activity.

More particularly, the present invention provides a bacterium which hasbeen modified to attenuate the expression of the genes sdaA, sdaB, tdcGand glyA.

The genes sdaA, sdaB and tdcG encode L-serine deaminase I (SdaA),L-serine deaminase II (SdaB) and L-serine deaminase III (TdcG),respectively, which are the three enzymes carrying out the sole step inthe pathway of L-serine degradation, converting serine into the basiccellular building block, pyruvate. Further information regarding sdaA,sdaB and tdcG of, e.g., Escherichia coli is available at EcoCyc(www.biocyc.org) under Accession numbers EG10930, EG11623 and G7624,respectively. Representative nucleotide sequences of sdaA, sdaB and tdcGare set forth in SEQ ID NOs: 1 to 3, respectively.

The gene glyA encodes serine hydroxymethyltransferase (GlyA) whichconverts serine to glycine, transferring a methyl group totetrahydrofolate, thus forming 5,10-methylene-tetrahydrofolate(5,10-mTHF). 5,10-mTHF is the major source of C1 units in the cell,making GlyA a key enzyme in the biosynthesis of purines, thymidine,methionine, choline and lipids. Further information regarding glyA of,e.g., Escherichia coli is available at EcoCyc (www.biocyc.org) underAccession number EG10408. A representative nucleotide sequence of glyAis set forth in SEQ ID NO: 4.

The expression of the genes may be attenuated by inactivation of thegenes. Thus, a bacterium according to the invention can be one which hasbeen modified to inactivate genes coding for polypeptides having serinedeaminase activity and to inactive a gene coding for a polypeptidehaving serine hydroxymethyltransferase activity. According to particularembodiments, the expression of the genes sdaA, sdaB, tdcG and glyA isattenuated by inactivation of these genes. Thus, a bacterium accordingto the invention can be one which has been modified to inactivate thegenes sdaA, sdaB, tdcG and glyA.

Expression of a gene can be attenuated by introducing a mutation intothe gene on the chromosome so that the intracellular activity of theprotein encoded by the gene is decreased as compared to an unmodifiedstrain. Mutations which result in attenuation of expression of the geneinclude the replacement of one nucleotide or more to cause an amino addsubstitution in the protein encoded by the gene (missense mutation),introduction of a stop codon (nonsense mutation), deletion or insertionof nucleotides to cause a frame shift, insertion of a drug-resistancegene, or deletion of a part of the gene or the entire gene (Qiu andGoodman, 1997; Kwon et al., 2000). Expression can also be attenuated bymodifying an expression regulating sequence such as the promoter, theShine-Dalgarno (SD) sequence, etc. (W095/34672).

For example, the following methods may be employed to introduce amutation by gene recombination. A mutant gene encoding a mutant proteinwith decreased activity can be prepared, and the bacterium to bemodified can be transformed with a DNA fragment containing the mutantgene. Then, the native gene on the chromosome is replaced with themutant gene by homologous recombination, and the resulting strain can beselected. Gene replacement using homologous recombination can beconducted by employing a linear DNA, which is known as “lambda-redmediated gene replacement” (Datsenko and Wanner, 2000), or by employinga plasmid containing a temperature-sensitive replication origin (U.S.Pat. No. 6,303,383 or JP 05-007491 A). Furthermore, site-specificmutation by gene substitution can also be incorporated using homologousrecombination such as set forth above using a plasmid which is unable toreplicate in the host.

Expression of the gene can also be attenuated by inserting a transposonor an IS factor into the coding region of the gene (U.S. Pat. No.5,175,107), or by conventional methods, such as by mutagenesis with UVirradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine),site-directed mutagenesis, gene disruption using homologousrecombination, and/or gene replacement (Yu et al., 2000; and Datsenkoand Wanner, 2000), such as the “lambda-red mediated gene replacement”.The lambda-red mediated gene replacement is a particularly suitablemethod to inactive one or more genes as described herein. Hence,according to particular embodiments, expression of genes is attenuatedby inactivation of the genes using lambda-red mediated gene replacement.

As shown in FIG. 3, the inactivation of all four genes (sdaA, sdaB, tdcGand glyA) involved in serine degradation results in the highest specificproductivity and the highest yield from glucose compared to inactivationof only the three genes involved in L-serine degradation via the serineto pyruvate pathway (sdaA, sdaB, and tdcG).

Serine is produced from glyceraldehyde-3-phosphate using three enzymesencoded by the genes serA (encoding a 3-phosphoglycerate dehydrogenase),serB (encoding a phosphoserine phosphatase) and serC (encoding aphosphoserine aminotransferase). In order to increase production ofL-serine, these genes may be overexpressed. Relevant informationregarding serA, serB and serC of, e.g., Escherichia coli is available atEcoCyc (www.biocyc.org) under Accession numbers EG10944, EG10945 andEG10946, respectively.

Therefore, according to certain embodiments, the bacterium has beenmodified to overexpress a 3-phosphoglycerate dehydrogenase, aphosphoserine phosphatase and a phosphoserine aminotransferase. Moreparticularly, the bacterium has been further modified to overexpress thegenes serA, serB and serC. This may be achieved by introducing into thebacterium one or more (such as two or three) exogenous nucleic acidmolecules, such as one or more vectors, comprising a nucleotide sequenceencoding a 3-phosphoglycerate dehydrogenase, a nucleotide sequenceencoding a phosphoserine phosphatase and/or a nucleotide sequenceencoding a phosphoserine aminotransferase.

The 3-phosphoglycerate dehydrogenase may be derived from the samespecies as the bacterium in which it is overexpressed or may be derivedfrom a species different to the one in which it is overexpressed (i.e.it is heterologous). According to certain embodiments, the3-phosphoglycerate dehydrogenase is derived from the same species as thebacterium in which it is overexpressed. According to certain otherembodiments, the 3-phosphoglycerate dehydrogenase is derived from aspecies different to the one in which it is overexpressed (i.e. it isheterologous).

According to certain embodiments, the bacterium comprises an exogenousnucleic acid molecule comprising a nucleotide sequence encoding a3-phosphoglycerate dehydrogenase. The exogenous nucleic acid moleculemay comprise a nucleotide sequence encoding a polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 5. The exogenous nucleicacid molecule may comprise a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set which has at least about 90%, atleast about 93%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 5. Preferably, thepolypeptide has 3-phosphoglycerate dehydrogenase activity. Morepreferably, the polypeptide has 3-phosphoglycerate dehydrogenaseactivity similar to that of the polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 5. The exogenous nucleic acid moleculemay comprise a nucleotide sequence encoding a polypeptide comprising anamino acid sequence set which has at least about 95%, such as at leastabout 96%, at least about 97%, at least about 98%, or at least about99%, sequence identity to the amino acid sequence set forth in SEQ IDNO: 5. Preferably, the polypeptide has 3-phosphoglycerate dehydrogenaseactivity. More, preferably, the polypeptide has 3-phosphoglyceratedehydrogenase activity similar to that of the polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 5. The exogenous nucleicacid molecule may comprise a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 5, wherein 1or more, such as about 1 to about 50, about 1 to about 40, about 1 toabout 35, about 1 to about 30, about 1 to about 25, about 1 to about 20,about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1to about 3, amino acid residues are substituted, deleted, and/orinserted. Preferably, the polypeptide has 3-phosphoglyceratedehydrogenase activity. More preferably, the polypeptide has3-phosphoglycerate dehydrogenase activity similar to that of thepolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 5.The exogenous nucleic acid molecule may comprise a nucleotide sequenceencoding a polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 5, wherein about 1 to about 5, such as about 1 to about 3,amino acid residues are substituted, deleted, and/or inserted.Preferably, the polypeptide has 3-phosphoglycerate dehydrogenaseactivity. More preferably, the polypeptide has 3-phosphoglyceratedehydrogenase activity similar to that of the polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 5.

It is further beneficial to overexpress a mutant serA gene which encodesa 3-phosphoglycerate dehydrogenase being resistant towards feedbackinhibition of serine. This may, for example, be achieved by deleting thelast four C-terminal residues of the wild type 3-phosphoglyceratedehydrogenase (SerA). Alternatively, feedback inhibition of SerA can beremoved by mutating the three residues H344, N346 and N364 to alanine. Arepresentative amino acid sequence of such 3-phosphoglyceratedehydrogenase mutant is set forth in SEQ ID NO: 6. Therefore, accordingto particular embodiments, the bacterium comprises an exogenous nucleicacid molecule comprising a nucleotide sequence encoding a3-phosphoglycerate dehydrogenase being resistant towards feedbackinhibition of serine. The exogenous nucleic acid molecule may comprise anucleotide sequence encoding a polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 6. The exogenous nucleic acid moleculemay comprise a nucleotide sequence encoding a polypeptide comprising anamino acid sequence set which has at least about 90%, at least about93%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99%, sequence identity to the aminoacid sequence set forth in SEQ ID NO: 6. Preferably, the polypeptide has3-phosphoglycerate dehydrogenase activity. More preferably, thepolypeptide has 3-phosphoglycerate dehydrogenase activity similar tothat of the polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 6. The exogenous nucleic acid molecule may comprise anucleotide sequence encoding a polypeptide comprising an amino acidsequence set which has at least about 95%, such as at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 6.Preferably, the polypeptide has 3-phosphoglycerate dehydrogenaseactivity. More preferably, the polypeptide has 3-phosphoglyceratedehydrogenase activity similar to that of the polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 6. The exogenous nucleicacid molecule may comprise a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 6, wherein 1or more, such as about 1 to about 50, about 1 to about 40, about 1 toabout 35, about 1 to about 30, about 1 to about 25, about 1 to about 20,about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1to about 3, amino acid residues are substituted, deleted, and/orinserted. Preferably, the polypeptide has 3-phosphoglyceratedehydrogenase activity. More preferably, the polypeptide has3-phosphoglycerate dehydrogenase activity similar to that of thepolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 6.The exogenous nucleic acid molecule may comprise a nucleotide sequenceencoding a polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 6, wherein about 1 to about 5, such as about 1 to about 3,amino acid residues are substituted, deleted, and/or inserted.Preferably, the polypeptide has 3-phosphoglycerate dehydrogenaseactivity. More preferably, the polypeptide has 3-phosphoglyceratedehydrogenase activity similar to that of the polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 6.

The phosphoserine phosphatase may be derived from the same species asthe bacterium in which it is overexpressed or may be derived from aspecies different to the one in which it is overexpressed (i.e. it isheterologous). According to certain embodiments, the phosphoserinephosphatase is derived from the same species as the bacterium in whichit is overexpressed.

According to certain other embodiments, the phosphoserine phosphatase isderived from a species different to the one in which it is overexpressed(i.e. it is heterologous).

According to certain embodiments, the bacterium comprises an exogenousnucleic acid molecule comprising a nucleotide sequence encoding aphosphoserine phosphatase. The exogenous nucleic acid molecule maycomprise a nucleotide sequence encoding a polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 7. The exogenous nucleicacid molecule may comprise a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set which has at least about 90%, atleast about 93%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 7. Preferably, thepolypeptide has phosphoserine phosphatase activity. More preferably, thepolypeptide has phosphoserine phosphatase activity similar to that ofthe polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 7. The exogenous nucleic acid molecule may comprise a nucleotidesequence encoding a polypeptide comprising an amino acid sequence setwhich has at least about 95%, such as at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 7. Preferably, thepolypeptide has phosphoserine phosphatase activity. More preferably, thepolypeptide has phosphoserine phosphatase activity similar to that ofthe polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 7. The exogenous nucleic acid molecule may comprise a nucleotidesequence encoding a polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 7, wherein 1 or more, such as about 1 to about 50,about 1 to about 40, about 1 to about 35, about 1 to about 30, about 1to about 25, about 1 to about 20, about 1 to about 15, about 1 to about10, about 1 to about 5, or about 1 to about 3, amino acid residues aresubstituted, deleted, and/or inserted. Preferably, the polypeptide hasphosphoserine phosphatase activity. More preferably, the polypeptide hasphosphoserine phosphatase activity similar to that of the polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 7. Theexogenous nucleic acid molecule may comprise a nucleotide sequenceencoding a polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 7, wherein about 1 to about 5, such as about 1 to about 3,amino acid residues are substituted, deleted, and/or inserted.Preferably, the polypeptide has phosphoserine phosphatase activity. Morepreferably, the polypeptide has phosphoserine phosphatase activitysimilar to that of the polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 7.

The phosphoserine aminotransferase may be derived from the same speciesas the bacterium in which it is overexpressed or may be derived from aspecies different to the one in which it is overexpressed (i.e. it isheterologous). According to certain embodiments, the phosphoserineaminotransferase is derived from the same species as the bacterium inwhich it is overexpressed. According to certain other embodiments, thephosphoserine aminotransferase is derived from a species different tothe one in which it is overexpressed (i.e. it is heterologous).

According to certain embodiments, the bacterium comprises an exogenousnucleic acid molecule comprising a nucleotide sequence encoding aphosphoserine aminotransferase. The exogenous nucleic acid molecule maycomprise a nucleotide sequence encoding a polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 8. The exogenous nucleicacid molecule may comprise a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set which has at least about 90%, atleast about 93%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 8. Preferably thepolypeptide has phosphoserine aminotransferase activity. Morepreferably, the polypeptide has phosphoserine aminotransferase activitysimilar to that of the polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 8. The exogenous nucleic acid molecule may comprisea nucleotide sequence encoding a polypeptide comprising an amino acidsequence set which has at least about 95%, such as at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 8.Preferably, the polypeptide has phosphoserine aminotransferase activity.More preferably, the polypeptide has phosphoserine aminotransferaseactivity similar to that of the polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 8. The exogenous nucleic acid moleculemay comprise a nucleotide sequence encoding a polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 8, wherein 1 or more, suchas about 1 to about 50, about 1 to about 40, about 1 to about 35, about1 to about 30, about 1 to about 25, about 1 to about 20, about 1 toabout 15, about 1 to about 10, about 1 to about 5, or about 1 to about3, amino acid residues are substituted, deleted, and/or inserted.Preferably, the polypeptide has phosphoserine aminotransferase activity.More preferably, the polypeptide has phosphoserine aminotransferaseactivity similar to that of the polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 8. The exogenous nucleic acid moleculemay comprise a nucleotide sequence encoding a polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 8, wherein about 1 to about5, such as about 1 to about 3, amino acid residues are substituted,deleted, and/or inserted. Preferably, the polypeptide has phosphoserineaminotransferase activity. More preferably, the polypeptide hasphosphoserine aminotransferase activity similar to that of thepolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 8.

A bacterium, such as Escherichia coli, which has been modified toattenuate expression of genes coding for polypeptides having serinedeaminase activity and to attenuate expression of a gene coding for apolypeptide having serine hydroxymethyltransferase (e.g., byinactivation of the genes), may show a low tolerance towards serine.Therefore, it would be desirable to provide a bacterium which showsincreased tolerance towards serine.

In this respect, the present inventors have found that product toxicitycan be reduced by overexpression of novel exporters, by evolvingbacterial strains by random mutagenesis, and by adaptive evolution. As aresult, bacteria having increased tolerance towards serine are provided.“Increased tolerance” as used herein means that a bacterium is capableof growing in a minimal culture medium (such as M9 minimal medium)comprising L-serine at a concentration of at least about 6.25 g/L.

According to certain embodiments, a bacterium of the present inventionis capable of growing in a minimal culture medium (such as M9 minimalmedium) comprising L-serine at a concentration of at least about 6.25g/L (such as at least about 12.5 g/L). According to particularembodiments, the bacterium is capable of growing in a minimal culturemedium (such as M9 minimal medium) comprising L-serine at aconcentration of at least about 12.5 g/L (such as at least about 25g/L). According to particular embodiments, the bacterium is capable ofgrowing in a minimal culture medium (such as M9 minimal medium)comprising L-serine at a concentration of at least about 25 g/L (such asat least about 40 g/L). According to particular embodiments, thebacterium is capable of growing in a minimal culture medium (such as M9minimal medium) comprising L-serine at a concentration of at least about40 g/L (such as at least about 50 g/L). According to particularembodiments, the bacterium is capable of growing in a minimal culturemedium (such as M9 minimal medium) comprising L-serine at aconcentration of at least about 50 g/L (such as at least about 75 g/L).According to particular embodiments, the bacterium is capable of growingin a minimal culture medium (such as M9 minimal medium) comprisingL-serine at a concentration of at least about 75 g/L (such as at leastabout 100 g/L. According to particular embodiments, the bacterium iscapable of growing in a minimal culture medium (such as M9 minimalmedium) comprising L-serine at a concentration of at least about 100g/L.

Preferably, the minimal culture medium, such as M9 minimal medium, issupplemented with 2 mM glycine and 2 g/L glucose. The bacterium isgenerally cultivated using adequate aeration at about 37° C. for aperiod of about 24 to about 40 hours.

According to certain embodiments, a bacterium of the present inventionis capable of growing in a minimal culture medium (such as M9 minimalmedium) comprising L-serine at a concentration of at least about 6.25g/L (such as at least about 12.5 g/L) at a growth rate of at least about0.1 hr⁻¹ during exponential growth. According to particular embodiments,a bacterium of the invention is capable of growing in a minimal culturemedium (such as M9 minimal medium) comprising L-serine at aconcentration of at least about 12.5 g/L (such as at least about 25 g/L)at a growth rate of at least about 0.1 hr⁻¹ during exponential growth.According to particular embodiments, a bacterium of the invention iscapable of growing in a minimal culture medium (such as M9 minimalmedium) comprising L-serine at a concentration of at least about 25 g/L(such as at least about 50 g/L) at a growth rate of at least about 0.1hr⁻¹ during exponential growth.

Preferably, the minimal culture medium, such as M9 minimal medium, issupplemented with 2 mM glycine and 2 g/L glucose. The bacterium isgenerally cultivated using adequate aeration at about 37° C. for aperiod of about 24 to about 40 hours.

One novel exporter which when overexpressed in a bacterium improvedtolerance towards serine is the O-acetylserine/cysteine export proteinencoded by the gene ydeD. Further information regarding ydeD of, e.g.,Escherichia coli is available at EcoCyc (www.biocyc.org) under Accessionnumbers EG11639. A representative amino acid sequence of such exporterprotein is set forth in SEQ ID NO: 9. Therefore, the present inventionprovides a bacterium which has been modified to overexpress the geneydeD.

According to certain embodiments, a bacterium of the invention comprisesan exogenous nucleic acid molecule, such as an expression vector,comprising a nucleotide sequence encoding the protein product of thegene ydeD. According to particular embodiments, the bacterium comprisesan exogenous nucleic acid molecule comprising a nucleotide sequenceencoding a polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 9. The exogenous nucleic acid molecule may comprise anucleotide sequence encoding a polypeptide comprising an amino acidsequence set which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 9. Preferably, the polypeptide has O-acetylserineand/or cysteine transporter activity. More preferably, the polypeptidehas O-acetylserine and/or cysteine transporter activity similar to thatof the polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 9. The exogenous nucleic acid molecule may comprise a nucleotidesequence encoding a polypeptide comprising an amino acid sequence setwhich has at least about 95%, such as at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 9. Preferably, thepolypeptide has O-acetylserine and/or cysteine transporter activity.More preferably, the polypeptide has O-acetylserine and/or cysteinetransporter activity similar to that of the polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 9. The exogenous nucleicacid molecule may comprise a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 9, wherein 1or more, such as about 1 to about 50, about 1 to about 40, about 1 toabout 35, about 1 to about 30, about 1 to about 25, about 1 to about 20,about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1to about 3, amino acid residues are substituted, deleted, and/orinserted. Preferably, the polypeptide has O-acetylserine and/or cysteinetransporter activity. More preferably, the polypeptide hasO-acetylserine and/or cysteine transporter activity similar to that ofthe polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 9. The exogenous nucleic acid molecule may comprise a nucleotidesequence encoding a polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 9, wherein about 1 to about 5, such as about 1 toabout 3, amino acid residues are substituted, deleted, and/or inserted.Preferably, the polypeptide has O-acetylserine and/or cysteinetransporter activity. More preferably, the polypeptide hasO-acetylserine and/or cysteine transporter activity similar to that ofthe polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 9.

A modified YdeD polypeptide containing an additional stretch of 6histidine residues at the C-terminus is set forth in SEQ ID NO: 10.According to particular embodiments, the bacterium comprises anexogenous nucleic acid molecule comprising a nucleotide sequenceencoding a polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 10. The exogenous nucleic acid molecule may comprise anucleotide sequence encoding a polypeptide comprising an amino acidsequence set which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 10. Preferably, the polypeptide has O-acetylserineand/or cysteine transporter activity. More preferably, the polypeptidehas O-acetylserine and/or cysteine transporter activity similar to thatof the polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 10. The exogenous nucleic acid molecule may comprise a nucleotidesequence encoding a polypeptide comprising an amino acid sequence setwhich has at least about 95%, such as at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 10. Preferably, thepolypeptide has O-acetylserine and/or cysteine transporter activity.More preferably, the polypeptide has O-acetylserine and/or cysteinetransporter activity similar to that of the polypeptide comprising anamino acid sequence set forth in SEQ ID NO: 10. The exogenous nucleicacid molecule may comprise a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 10, wherein 1or more, such as about 1 to about 50, about 1 to about 40, about 1 toabout 35, about 1 to about 30, about 1 to about 25, about 1 to about 20,about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1to about 3, amino acid residues are substituted, deleted, and/orinserted. Preferably, the polypeptide has O-acetylserine and/or cysteinetransporter activity. More preferably, the polypeptide hasO-acetylserine and/or cysteine transporter activity similar to that ofthe polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 10. The exogenous nucleic acid molecule may comprise a nucleotidesequence encoding a polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 10, wherein about 1 to about 5, such as about 1 toabout 3, amino acid residues are substituted, deleted, and/or inserted.Preferably, the polypeptide has O-acetylserine and/or cysteinetransporter activity. More preferably, the polypeptide hasO-acetylserine and/or cysteine transporter activity similar to that ofthe polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 10.

As shown in FIG. 4, growth of a bacterium, such as E. coli, lacking themain serine degradation pathways is severely growth inhibited in thepresence of even low concentrations of serine. Upon overexpression ofydeD, the tolerance towards serine is increased substantially,suggesting that YdeD may potentially transport serine out of the cell.

A bacterium of the invention having improved tolerance towards serine,such as one capable of growing in a minimal culture medium comprisingL-serine at a concentration of at least about 6.25 g/L as mentionedabove, can be obtained by random mutagenesis or by adaptive evolution.Respective details are provided in Examples 4 and 5, respectively.

Adaptive evolution may, for example, be achieved by carrying out thefollowing method: Prior to the start of the experiment, suitable tubesare filled with 25 ml of culture media which are kept at 37° C. in aheat block. Controlled aeration is obtained using magnetic tumblestirrers placed inside the tubes and spinning at 1,800 rpm. At the startof the experiment, a single colony (of the starter strain) is grownovernight in one of the tubes, and 100 μL aliquots are used to inoculatea new tube containing 25 ml of fresh culture media. As the bacteriagrow, multiple OD measurements at 600 nm are performed. Growth rates arecalculated by taking the slope of a least-square linear regression linefit to the logarithm of the OD measurements. Once reaching a target ODof 0.4, 100 μl of culture are used to inoculate a new tube containing 25ml of culture media. This way, cultures are serially passed (2-3 timesper day) to tubes with fresh media after reaching the targeted celldensity such that stationary phase is never reached. The experiment isinitiated with an L-serine concentration of 3 g/L serine, followed by anincrease to 6 g/L of L-Serine after the desired growth rate has beenreached. Once the populations achieved a stable phenotype (i.e. growthrate), the L-Serine concentration is increased to 12 g/L. This processis repeated iteratively using 24, 50, 75, and 100 g/L of L-serine. Thefinal population may then be plated on the LB-agar for furthercultivation and selection of an L-serine tolerant strain. The foregoingmethod may be performed manually or by using an automated system enablethe propagation of evolving populations over the course of many dayswhile monitoring their growth rates.

As further demonstrate herein, the present inventors have identifiedbeneficial mutations in a number of genes which confer tolerance towardsL-serine. Respective genes and mutations are depicted in Table 55.

One such gene is thrA which encodes an aspartate kinase I/homoserinedehydrogenase I (ThrA). Further information regarding thrA of, e.g.,Escherichia coli is available at EcoCyc (www.biocyc.org) under Accessionnumbers EG10998. A representative amino acid sequence of a wild typeAspartate kinase I/homoserine dehydrogenase I (ThrA) is set forth in SEQID NO: 11. As demonstrated in Example 6 and 10, introducing certainmutation within the amino acid sequence of the aspartate kinaseI/homoserine dehydrogenase results in a very significant increase intolerance towards serine (FIGS. 7 and 11). Particularly, the followingmutations have been shown to be beneficial: Y356C, S357R and S359R.

According to certain embodiments, the present invention provides abacterium which expresses an aspartate kinase I/homoserine dehydrogenaseI (ThrA) mutant which is not inhibited by L-serine.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the thrA gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions in theencoded polypeptide at a position selected from the group consisting ofY356, S357 and S359. A bacterium of the invention may thus express anaspartate kinase I/homoserine dehydrogenase I (ThrA) having one or moreamino acid substitutions at a position selected from the groupconsisting of Y356, S357 and S359. More particularly, a bacterium of theinvention may express a polypeptide having the amino acid sequence setforth in SEQ ID NO: 11 which comprises one or more amino acidsubstitutions at a position selected from the group consisting of Y356,S357 and S359. Preferably, the one or more amino acid substitutions arenon-conservative substitutions.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position Y356. A bacterium of the invention may thusexpress a polypeptide encoded by the thrA gene, wherein said polypeptidecomprises an amino add substitution at position Y356. According toparticular embodiments, the amino acid substitution is selected from thegroup consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G,Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R, andY356L. According to other particular embodiments, the amino acidsubstitution is selected from the group consisting of Y356C, Y356T,Y356V, Y356W, Y356Q, Y356G, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H,Y356R, and Y356L. According to more particular embodiments, the aminoacid substitution is selected from the group consisting of Y356C, Y356T,Y356W, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R, and Y356L. According toother more particular embodiments, the amino acid substitution isselected from the group consisting of Y356C, Y356W, Y356F, Y356I, Y356P,Y356R, and Y356L.

According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a Y356Csubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a Y356C substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a Y356T substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa Y356T substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a Y356V substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a Y356V substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a Y356Ssubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a Y356S substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a Y356W substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa Y356W substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a Y356G substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a Y356G substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a Y356Nsubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a Y356N substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a Y356D substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa Y356D substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a Y356E substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a Y356E substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a Y356Fsubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a Y356F substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a Y356A substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa Y356I substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a Y356A substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a Y356I substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a Y356Psubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a Y356P substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a Y356H substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa Y356H substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a Y356R substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a Y356R substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a Y356Lsubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a Y356L substitution.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position S357. A bacterium of the invention may thusexpress a polypeptide encoded by the thrA gene, wherein said polypeptidecomprises an amino acid substitution at position S357. According toparticular embodiments, the amino acid substitution is selected from thegroup consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A,S357N, S357F, S357H, S357K, S357I and S357M. According to otherparticular embodiments, the amino acid substitution is selected from thegroup consisting of S357R, 357V, S357G, 357L, S357Y, S357A, S357N, S357Fand S357H. According to more particular embodiments, the amino acidsubstitution is selected from the group consisting of S357R, S357A,S357N and S357F. According to other more particular embodiments, theamino acid substitution is selected from the group consisting of S357Aand S357F.

According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a S357Rsubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a S357R substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a S357V substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa S357V substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a S357P substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a S357P substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a S357Gsubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a S357G substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a S357L substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa S357L substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a S357Y substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a S357Y substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a S357Asubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a S357A substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a S357N substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa S357N substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a S357F substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a S357F substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a S357Hsubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a S357H substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a S357K substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa S357K substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a S357I substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a S357I substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a S357Msubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a S357M substitution.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position S359. A bacterium of the invention may thusexpress a polypeptide encoded by the thrA gene, wherein said polypeptidecomprises an amino acid substitution at position S359. According toparticular embodiments, the amino acid substitution is selected from thegroup consisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V,S359Q, S359A, S359C, S359K, S359E and S359L. According to otherparticular embodiments, the amino acid substitution is selected from thegroup consisting of S359R, S359F, S359T, S359P, S359V, S359Q, S359A,S359C, S359K, S359E and S359L. According to more particular embodiments,the amino acid substitution is selected from the group consisting ofS359R, S359T, S359P, S359V, S359Q, S359A, S359E and S359L. According toother more particular embodiments, the amino acid substitution isselected from the group consisting of S359R, S359T, S359P, S3590, S359A,S359E and S359L. According to other more particular embodiments, theamino acid substitution is selected from the group consisting of S359R,S359T, S359Q, S359A and S359E. According to other more particularembodiments, the amino acid substitution is selected from the groupconsisting of S359R, S359T and S359A. According to other more particularembodiments, the amino acid substitution is selected from the groupconsisting of S359R and S359A.

According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a S359Rsubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a S359R substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a S359G substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa S359G substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a S359M substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a S359M substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a S359Fsubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a S359F substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a S359T substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa S359T substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a S359P substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a S359P substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a S359Vsubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a S359V substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a S359Q substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa S359Q substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a S359A substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a S359A substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a S359Csubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a S359C substitution. According to certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a S359K substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa S359K substitution. According to certain embodiments, the bacteriumcomprises within the thrA gene one or more nucleotide substitutionsresulting in a S359E substitution in the encoded polypeptide. Abacterium of the invention may thus express a polypeptide encoded by thethrA gene, wherein said polypeptide comprises a S359E substitution.According to certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a S359Lsubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a S359L substitution.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position Y356, one or more nucleotide substitutionsresulting in an amino acid substitution in the encoded polypeptide atposition S357 and/or one or more nucleotide substitutions resulting inan amino acid substitution in the encoded polypeptide at position S359;wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N,Y3560, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; thesubstitution at position S357 is selected from the group consisting ofS357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S3590Q S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position Y356 and/or one or more nucleotide substitutionsresulting in an amino acid substitution in the encoded polypeptide atposition S357; wherein the substitution at position Y356 is selectedfrom the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356QY356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R andY356L; and the substitution at position S357 is selected from the groupconsisting of S357R, S357V, S357P, S357G, S357I, S357Y, S357A, S357N,S357F, S357H, S357K, S357I and S357M.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position Y356 and/or one or more nucleotide substitutionsresulting in an amino acid substitution in the encoded polypeptide atposition S359; wherein the substitution at position Y356 is selectedfrom the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y3560,Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R andY356L; and the substitution at position S359 is selected from the groupconsisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q,S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position S357 and/or one or more nucleotide substitutionsresulting in an amino acid substitution in the encoded polypeptide atposition S359; wherein the substitution at position S357 is selectedfrom the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y,S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitutionat position S359 is selected from the group consisting of S359R, S359G,S359M, S359F, S359T, S359P, S359V, S3590Q S359A, S359C, S359K, S359E andS359L.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position Y356, one or more nucleotide substitutionsresulting in an amino acid substitution in the encoded polypeptide atposition S357 and one or more nucleotide substitutions resulting in anamino acid substitution in the encoded polypeptide at position S359;wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N,Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; thesubstitution at position S357 is selected from the group consisting ofS357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S359a, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position Y356 and one or more nucleotide substitutionsresulting in an amino acid substitution in the encoded polypeptide atposition S357; wherein the substitution at position Y356 is selectedfrom the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q,Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R andY356L; and the substitution at position S357 is selected from the groupconsisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N,S357F, S357H, S357K, S357I and S357M.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position Y356 and one or more nucleotide substitutionsresulting in an amino acid substitution in the encoded polypeptide atposition S359; wherein the substitution at position Y356 is selectedfrom the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q,Y356G, Y356N, Y356D, Y356E, Y356F and Y356A; and the substitution atposition S359 is selected from the group consisting of S359R, S359G,S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E andS359L.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position S357 and one or more nucleotide substitutionsresulting in an amino acid substitution in the encoded polypeptide atposition S359; wherein the substitution at position S357 is selectedfrom the group consisting of S357R, S357V, S357P, S357G, S357L, S357Y,S357A, S357N, S357F, S357H, S357K, S357I and S357M; and the substitutionat position S359 is selected from the group consisting of S359R, S359G,S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E andS359L.

According to certain embodiments, a bacterium of the invention express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position Y356, S357 and/orS359, wherein the substitution at position Y356 is selected from thegroup consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G,Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L;the substitution at position S357 is selected from the group consistingof S357R, S357V, S357P, S357G, S357I, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position Y356 and/or S357,wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N,Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; andthe substitution at position S357 is selected from the group consistingof S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M.

According to certain embodiments, a bacterium of the invention express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position Y356 and/or S359,wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N,Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; andthe substitution at position S359 is selected from the group consistingof S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C,S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position S357 and/or S359,wherein the substitution at position S357 is selected from the groupconsisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N,S357F, S357H, S357K, S357I and S357M; and the substitution at positionS359 is selected from the group consisting of S359R, S359G, S359M,S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position Y356, wherein thesubstitution at position Y356 is selected from the group consisting ofY356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E,Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L.

According to certain embodiments, a bacterium of the invention express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position S357, wherein thesubstitution at position S357 is selected from the group consisting ofS357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position S359, wherein thesubstitution at position S359 is selected from the group consisting ofS359R, S359G, S359M, S359F, S359T, S359P, S359V, S3590, S359A, S359C,S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position Y356 and S357,wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356 Y356G, Y356N,Y356D, Y356E, Y356F and Y356A; and the substitution at position S357 isselected from the group consisting of S357R, S357V, S357P, S357G, S357L,S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M.

According to certain embodiments, a bacterium of the invention express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position Y356 and S359,wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N,Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; andthe substitution at position S359 is selected from the group consistingof S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C,S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position S357 and S359,wherein the substitution at position S357 is selected from the groupconsisting of S357R, S357V, S357P, S357G, S357I, S357Y, S357A, S357N,S357F, S357H, S357K, S357I and S357M; and the substitution at positionS359 is selected from the group consisting of S359R, S359G, S359M,S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position Y356, S357 andS359, wherein the substitution at position Y356 is selected from thegroup consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G,Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L;the substitution at position S357 is selected from the group consistingof S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by cysteine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by threonine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by valine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by serine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by tryptophan. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by glutamine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by glycine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by asparagine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by aspartic acid. Accordingto certain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by glutamic acid. Accordingto certain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by phenylalanine. Accordingto certain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by alanine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by isoleucine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by proline. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by histidine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by arginine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 356 tyrosine is replaced by leucine.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by arginine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by valine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by proline. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by glycine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by leucine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by tyrosine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by alanine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by asparagine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by phenylalanine. Accordingto certain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by histidine.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by lysine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by isoleucine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 357 serine is replaced by methionine.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by arginine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by glycine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by methionine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by phenylalanine. Accordingto certain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by threonine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by proline. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by valine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by glutamine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by alanine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by cysteine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by lysine. According tocertain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by glutamic acid. Accordingto certain embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11,wherein at position 359 serine is replaced by leucine.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about90%, such at least about 93%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position Y356, S357 and/or S359,wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356, Y56N,Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; thesubstitution at position S357 is selected from the group consisting ofS357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about90%, such as at least about 93%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position Y356 and/or S357,wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N,Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; andthe substitution at position S357 is selected from the group consistingof S357R, S357V, S357P, S357G, S357I, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about90%, such as at least about 93%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position Y356 and/or S359,wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y3560, Y356G, Y356N,Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; andthe substitution at position S359 is selected from the group consistingof S359R, S359G, S359M, S359F, S359T, S359P, S359V, S3590, S359A, S359C,S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about90%, such as at least about 93%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position S357 and/or S359,wherein the substitution at position S357 is selected from the groupconsisting of S357R, S357V, S357P, S357G, S357I, S357Y, S357A, S357N,S357F, S357H, S357K, S357I and S357M; and the substitution at positionS359 is selected from the group consisting of S359R, S359G, S359M,S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about90%, such at least about 93%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position Y356, wherein thesubstitution at position Y356 is selected from the group consisting ofY356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E,Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about90%, such as at least about 93%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position S357, wherein thesubstitution at position S357 is selected from the group consisting ofS357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about90%, such as at least about 93%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position S359, wherein thesubstitution at position S359 is selected from the group consisting ofS359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q. S359A, S359C,S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about93%, such as at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 11 which comprises an aminoacid substitution at position Y356, wherein the substitution at positionY356 is selected from the group consisting of Y356C, Y356T, Y356V,Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I,Y356P, Y356H, Y356R and Y356L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about93%, such as at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 11 which comprises an aminoacid substitution at position S357, wherein the substitution at positionS357 is selected from the group consisting of S357R, S357V, S357P,S357G, S357I. S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about93%, such as at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 11 which comprises an aminoacid substitution at position S359, wherein the substitution at positionS359 is selected from the group consisting of S359R, S359G, S359M,S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about95%, such as at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 11 which comprises an amino acid substitution atposition Y356, wherein the substitution at position Y356 is selectedfrom the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q,Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R andY356L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about95%, such as at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 11 which comprises an amino acid substitution atposition S357, wherein the substitution at position S357 is selectedfrom the group consisting of S357R, S357V, S357P, S357G, S357I, S357Y,S357A, S357N, S357F, S357H, S357K, S357I and S357M.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about95%, such as at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 11 which comprises an amino acid substitution atposition S359, wherein the substitution at position S359 is selectedfrom the group consisting of S359R, S359G, S359M, S359F, S359T, S359P,S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about90%, such as at least about 93%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position Y356, S357 and S359,wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N,Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; thesubstitution at position S357 is selected from the group consisting ofS357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about90%, such as at least about 93%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position Y356 and S357, whereinthe substitution at position Y356 is selected from the group consistingof Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E,Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; and the substitutionat position S357 is selected from the group consisting of S357R, S357V,S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I andS357M.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about90%, such as at least about 93%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position Y356 and S359, whereinthe substitution at position Y356 is selected from the group consistingof Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E,Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; and the substitutionat position S359 is selected from the group consisting of S359R, S359G,S359M, S359F, S359T, S359P, S359V, S3590, S359A, S359C, S359K, S359E andS359L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence which has at least about90%, such as at least about 93%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 11 whichcomprises an amino acid substitution at position S357 and S359, whereinthe substitution at position Y356 is selected from the group consistingof Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E,Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; the substitution atposition S357 is selected from the group consisting of S357R, S357V,S357P, S357G, S357I, S357Y, S357A, S357N, S357F, S357H, S357K, S357I andS357M; and the substitution at position S359 is selected from the groupconsisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q,S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention expressesa polypeptide having an amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position Y356, S357 and/orS359, wherein the substitution at position Y356 is selected from thegroup consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G,Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L;the substitution at position S357 is selected from the group consistingof S357R, S357V, S357P, S357G, S357I, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S3590, S359A, S359C, S359K, S359E and S359L; and wherein 1or more, such as about 1 to about 50, about 1 to about 40, about 1 toabout 35, about 1 to about 30, about 1 to about 25, about 1 to about 20,about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1to about 3, further amino acid residues are substituted, deleted, and/orinserted.

The bacterium may express a polypeptide having an amino acid sequenceset forth in SEQ ID NO: 11 which comprises an amino acid substitution atposition Y356, S357 and/or S359, wherein the substitution at positionY356 is selected from the group consisting of Y356C, Y356T, Y356V,Y356S, Y356W, Y3560, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I,Y356P, Y356H, Y356R and Y356L; the substitution at position S357 isselected from the group consisting of S357R, S357V, S357P, S357G, S357L,S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; and thesubstitution at position S359 is selected from the group consisting ofS359R, S359G, S359M, S359F, S359T, S359P, S359V, S3590, S359A, S359C,S359K, S359E and S359L; and wherein about 1 to about 5, such as about 1to about 3, further amino acid residues are substituted, deleted, and/orinserted.

According to certain embodiments, the present invention provides abacterium which comprises within the thrA gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions in theencoded polypeptide selected from the group consisting of Y356C, S357Rand S359R. A bacterium of the invention may thus express a polypeptideencoded by the thrA gene, wherein said polypeptide comprises one or more(such as two or three) amino acid substitutions selected from the groupconsisting of Y356C, S357R and S359R. According to certain embodiments,a bacterium of the invention comprises within the thrA gene one or morenucleotide substitutions resulting in one or more (such as two) aminoacid substitutions in the encoded polypeptide selected from the groupconsisting of Y356C and S357R. A bacterium of the invention may thusexpress a polypeptide encoded by the thrA gene, wherein said polypeptidecomprises one or more (such as two) amino acid substitutions in theencoded polypeptide selected from the group consisting of Y356C andS357R. According to certain embodiments, a bacterium of the inventioncomprises within the thrA gene one or more nucleotide substitutionsresulting in one or more (such as two) amino acid substitutions in theencoded polypeptide selected from the group consisting of Y356C andS359R. A bacterium of the invention may thus express a polypeptideencoded by the thrA gene, wherein said polypeptide comprises one or more(such as two) amino acid substitutions in the encoded polypeptideselected from the group consisting of Y356C and S359R. According tocertain embodiments, a bacterium of the invention comprises within thethrA gene one or more nucleotide substitutions resulting in one or more(such as two) amino acid substitutions in the encoded polypeptideselected from the group consisting of S357R and S359R. A bacterium ofthe invention may thus express a polypeptide encoded by the thrA gene,wherein said polypeptide comprises one or more (such as two) amino acidsubstitutions in the encoded polypeptide selected from the groupconsisting of S357R and S359R.

According the certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a Y356Csubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a Y356C substitution. According the certainembodiments, the bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in a S357R substitution in theencoded polypeptide. A bacterium of the invention may thus express apolypeptide encoded by the thrA gene, wherein said polypeptide comprisesa S357R substitution.

According the certain embodiments, the bacterium comprises within thethrA gene one or more nucleotide substitutions resulting in a S359Rsubstitution in the encoded polypeptide. A bacterium of the inventionmay thus express a polypeptide encoded by the thrA gene, wherein saidpolypeptide comprises a S359R substitution.

A bacterium of the invention may thus express an aspartate kinaseI/homoserine dehydrogenase I (ThrA) having one or more amino acidsubstitutions selected from the group consisting of Y356C, S357R andS359R. More particularly, a bacterium of the invention may express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises one or more amino acid substitutions selected from thegroup consisting of Y356C, S357R and S359R. According to certainembodiments, the bacterium expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 11, wherein at position 356 tyrosine isreplaced by cysteine. According to certain embodiments, the bacteriumexpresses a polypeptide having the amino acid sequence set forth in SEQID NO: 11, wherein at position 357 serine is replaced by arginine.According to certain embodiments, the bacterium expresses a polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 11, wherein atposition 359 serine is replaced by arginine.

According to certain embodiments, the bacterium expresses a polypeptidehaving an amino acid sequence set which has at least about 90%, at leastabout 93%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 11 which comprises one ormore amino acid substitutions selected from the group consisting ofY356C, S357R and S359R.

According to certain embodiments, the bacterium expresses a polypeptidehaving an amino acid sequence which has at least about 90%, at leastabout 93%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 11, wherein at position 356tyrosine is replaced by cysteine. According to particular embodiments,the bacterium expresses a polypeptide having an amino acid sequencewhich has at least about 95%, such as at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 11, wherein at position 356tyrosine is replaced by cysteine.

According to certain embodiments, the bacterium expresses a polypeptidehaving the amino acid sequence which has at least about 90%, at leastabout 93%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 11, wherein at position 357serine is replaced by arginine. According to particular embodiments, thebacterium expresses a polypeptide having the amino acid sequence whichhas at least about 95%, such as at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 11, wherein at position 357serine is replaced by arginine.

According to certain embodiments, the bacterium expresses a polypeptidehaving the amino add sequence which has at least about 90%, at leastabout 93%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence Identity to theamino acid sequence set forth in SEQ ID NO: 11, wherein at position 359serine is replaced by arginine. According to particular embodiments, thebacterium expresses a polypeptide having the amino acid sequence whichhas at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 11, wherein at position 359 serine isreplaced by arginine.

According to certain embodiments, the bacterium expresses a polypeptidehaving an amino add sequence set forth in SEQ ID NO: 11 which comprisesone or more amino acid substitutions selected from the group consistingof Y356C, S357R and S359R, wherein 1 or more, such as about 1 to about50, about 1 to about 40, about 1 to about 35, about 1 to about 30, about1 to about 25, about 1 to about 20, about 1 to about 15, about 1 toabout 10, about 1 to about 5, or about 1 to about 3, further amino acidresidues are substituted, deleted, and/or inserted. The bacterium mayexpress a polypeptide having an amino acid sequence set forth in SEQ IDNO: 11 which comprises one or more amino acid substitutions selectedfrom the group consisting of Y356C, S357R and S359R, wherein about 1 toabout 5, or about 1 to about 3, further amino acid residues aresubstituted, deleted, and/or inserted.

The ThrA polypeptide mutant(s) described above may be (over-)expressedby the bacterium by way of an exogenous nucleic acid molecule, such asan expression vector, which has been introduced into the bacterium.Therefore, according to certain embodiments, the bacterium of theinvention comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding a aspartate kinase I/homoserinedehydrogenase I (ThrA) polypeptide mutant as described above.

For example, a bacterium of the invention may comprise an exogenousnucleic acid molecule, such as an expression vector, comprising anucleotide sequence encoding a polypeptide having the amino acidsequence set forth in SEQ ID NO: 11 which comprises an amino acidsubstitution at position Y356, S357 and/or S359. According to certainembodiments, a bacterium of the invention thus comprises an exogenousnucleic acid molecule comprising a nucleotide sequence encoding apolypeptide having the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position Y356, S357 and/orS359, wherein the substitution at position Y356 is selected from thegroup consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G,Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L;the substitution at position S357 is selected from the group consistingof S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention comprisesan exogenous nucleic acid molecule comprising a nucleotide sequenceencoding a polypeptide having an amino acid sequence which has at leastabout 90%, such at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 11which comprises an amino acid substitution at position Y356, S357 and/orS359. According to certain embodiments, a bacterium of the inventioncomprises an exogenous nucleic acid molecule comprising a nucleotidesequence encoding a polypeptide having an amino acid sequence which hasat least about 90%, such at least about 93%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity to the amino acid sequence set forth in SEQID NO: 11 which comprises an amino acid substitution at position Y356,S357 and/or S359, wherein the substitution at position Y356 is selectedfrom the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q,Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R andY356L; the substitution at position S357 is selected from the groupconsisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N,S357F, S357H, S357K, S357I and S357M; and the substitution at positionS359 is selected from the group consisting of S359R, S359G, S359M,S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention comprisesan exogenous nucleic acid molecule comprising a nucleotide sequenceencoding a polypeptide having an amino acid sequence which has at leastabout 93%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 11 which comprises an aminoacid substitution at position Y356, S357 and/or S359. According tocertain embodiments, a bacterium of the invention comprises an exogenousnucleic acid molecule comprising a nucleotide sequence encoding apolypeptide having an amino acid sequence which has at least about 93%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99%, sequence identity to the amino acidsequence set forth in SEQ ID NO: 11 which comprises an amino acidsubstitution at position Y356, S357 and/or S359, wherein thesubstitution at position Y356 is selected from the group consisting ofY356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E,Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; the substitution atposition S357 is selected from the group consisting of S357R, S357,S357P, S357357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357Iand S357M; and the substitution at position S359 is selected from thegroup consisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V,S359Q, S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, a bacterium of the invention comprisesan exogenous nucleic acid molecule comprising a nucleotide sequenceencoding a polypeptide having an amino acid sequence which has at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 11 which comprises an amino acid substitution atposition Y356, S357 and/or S359. According to certain embodiments, abacterium of the invention comprises an exogenous nucleic acid moleculecomprising a nucleotide sequence encoding a polypeptide having an aminoacid sequence which has at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least about 99%, sequence identityto the amino acid sequence set forth in SEQ ID NO: 11 which comprises anamino acid substitution at position Y356, S357 and/or S359, wherein thesubstitution at position Y356 is selected from the group consisting ofY356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E,Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; the substitution atposition S357 is selected from the group consisting of S357R, S357V,S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I andS357M; and the substitution at position S359 is selected from the groupconsisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q,S359A, S359C, S359K, S359E and S359L.

In this connection, the present invention further provides a (isolated)nucleic acid molecule, such an expression vector, comprising anucleotide sequence encoding a ThrA mutant as described above. Suchnucleic acid may be introduced into the bacterium of the invention.According to certain embodiments, the nucleic acid molecule comprises anucleotide sequence encoding a polypeptide having an amino acid sequencewhich has at least about 90%, such as at least about 93%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99%, sequence identity to the amino acid sequence set forthin SEQ ID NO: 11 which comprises an amino acid substitution at positionY356, S357 and/or S359. According to certain embodiments, the nucleicadd molecule comprises a nucleotide sequence encoding a polypeptidehaving an amino acid sequence which has at least about 90%, such as atleast about 93%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 11 which comprises an aminoacid substitution at position Y356, S357 and/or S359; wherein thesubstitution at position Y356 is selected from the group consisting ofY356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E,Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; the substitution atposition S357 is selected from the group consisting of S357R, S357V,S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I andS357M; and the substitution at position S359 is selected from the groupconsisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q,S359A, S359C, S359K, S359E and S359L.

According to certain embodiments, the nucleic acid molecule comprising anucleotide sequence encoding a polypeptide having the amino acidsequence set forth in SEQ ID NO: 11 which comprises an amino acidsubstitution at position Y356, S357 and/or S359. According to certainembodiments, the nucleic acid molecule comprising a nucleotide sequenceencoding a polypeptide having the amino acid sequence set forth in SEQID NO: 11 which comprises an amino acid substitution at position Y356,S357 and/or S359, wherein the substitution at position Y356 is selectedfrom the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q,Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R andY356L; the substitution at position S357 is selected from the groupconsisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N,S357F, S357H, S357K, S357I and S357M; and the substitution at positionS359 s selected from the group consisting of S359R, S359G, S359M, S359F,S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.

The (isolated) nucleic acid molecule may be the exogenous nucleic acidas detailed above. The (isolated) nucleic acid molecule may comprisesuitable regulatory elements such as a promoter that is functional inthe bacterial cell to cause the production of an mRNA molecule and thatIs operably linked to the nucleotide sequence encoding said polypeptide.Further details on suitable regulatory elements are provided below withrespect to an “exogenous” nucleic acid molecule, and apply mutatismutondis.

The present invention also provides a bacterium which comprises withinthe Irp gene one or more nucleotide substitutions resulting in the aminoacid substitution D143G in the encoded polypeptide. Further informationregarding Irp of, e.g., Escherichia coli is available at EcoCyc(www.biocyc.org) under Accession number EG10547. According to certainembodiments, a bacterium of the invention expresses a polypeptideencoded by the Irp gene, wherein in said polypeptide at position 143 Dis replaced by G. A representative amino acid sequence of a polypeptideencoded by the Irp gene is set forth in SEQ ID NO: 12. According toparticular embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 12 ora polypeptide having the amino acid sequence which has at least about90%, at least about 93%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 12, whereinin said amino acid sequence at position 143 D is replaced by G.

According to certain embodiments, the present invention provides abacterium which comprises within the Irp gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the Irp gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position D143. According to particular embodiments, abacterium of the invention expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 12 or a polypeptide having the aminoacid sequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 12, wherein in said amino acid sequence at position143 D is replaced by another amino acid. Preferably, the one or moreamino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium which comprises withinthe rho gene one or more nucleotide substitutions resulting in the aminoacid substitution R87L in the encoded polypeptide. Further informationregarding rho of, e.g., Escherichia coli such as nucleotide sequence ofthe gene or amino acid sequence of the encoded polypeptide is availableat EcoCyc (www.biocyc.org) under Accession number EG10845. According tocertain embodiments, a bacterium of the invention expresses apolypeptide encoded by the rho gene, wherein in said polypeptide atposition 87 R is replaced by L A representative amino acid sequence of apolypeptide encoded by the rho gene is set forth in SEQ ID NO: 13.According to particular embodiments, a bacterium of the inventionexpresses a polypeptide having the amino acid sequence set forth in SEQID NO: 13 or a polypeptide having the amino acid sequence which has atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 13,wherein in said amino acid sequence at position 87 R is replaced by L.

According to certain embodiments, the present invention provides abacterium which comprises within the rho gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the rho gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position R87. According to particular embodiments, abacterium of the invention expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 13 or a polypeptide having the aminoacid sequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 13, wherein in said amino acid sequence at position87 R is replaced by another amino acid. Preferably, the one or moreamino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium which comprises withinthe eno gene one or more nucleotide substitutions resulting in the aminoadd substitution V164L in the encoded polypeptide. Further informationregarding eno of, e.g., Escherichia coli is available at EcoCyc(www.biocyc.org) under Accession number EG10258. According to certainembodiments, a bacterium of the invention expresses a polypeptideencoded by the eno gene, wherein in said polypeptide at position 164 Vis replaced by L. A representative amino acid sequence of a polypeptideencoded by the eno gene is set forth in SEQ ID NO: 14. According toparticular embodiments, a bacterium of the invention may thus express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 14 ora polypeptide having the amino acid sequence which has at least about90%, at least about 93%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 14, whereinin said amino acid sequence at position at position 164 V is replaced byL.

According to certain embodiments, the present Invention provides abacterium which comprises within the eno gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the eno gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position V164. According to particular embodiments, abacterium of the invention expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 14 or a polypeptide having the aminoacid sequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 14, wherein in said amino acid sequence at position164 V is replaced by another amino acid. Preferably, the one or moreamino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium which comprises withinthe argP gene one or more nucleotide substitutions resulting in theamino acid substitution V164L in the encoded polypeptide. Furtherinformation regarding argP of, e.g., Escherichia coli is available atEcoCyc (www.biocyc.org) under Accession number EG10490. According tocertain embodiments, a bacterium of the invention expresses apolypeptide encoded by the argP gene, wherein in said polypeptide atposition 132 Q is replaced by K. A representative amino acid sequence ofa polypeptide encoded by the argP gene is set forth in SEQ ID NO: 15.According to particular embodiments, a bacterium of the inventionexpresses a polypeptide having the amino acid sequence set forth in SEQID NO: 15 or a polypeptide having the amino acid sequence which has atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 15,wherein in said amino acid sequence at position at position 132 Q isreplaced by K.

According to certain embodiments, the present invention provides abacterium which comprises within the argP gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the argP gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position Q132. According to particular embodiments, abacterium of the invention expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 15 or a polypeptide having the aminoacid sequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 15, wherein in said amino acid sequence at position132 Q is replaced by another amino acid. Preferably, the one or moreamino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium which comprises withinthe tufA gene one or more nucleotide substitutions resulting in theamino acid substitution G19V in the encoded polypeptide. Furtherinformation regarding tufA of, e.g., Escherichia coli is available atEcoCyc (www.biocyc.org) under Accession number EG11036. According tocertain embodiments, a bacterium of the invention expresses apolypeptide encoded by the tufA gene, wherein in said polypeptide atposition 19 G is replaced by V. A representative amino acid sequence ofa polypeptide encoded by the tufA gene is set forth in SEQ ID NO: 16.According to particular embodiments, a bacterium of the inventionexpresses a polypeptide having the amino acid sequence set forth in SEQID NO: 16 or a polypeptide having the amino acid sequence which has atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 16,wherein in said amino acid sequence at position at position 19 G isreplaced by V.

According to certain embodiments, the present invention provides abacterium which comprises within the tufA gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the tufA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position G19. According to particular embodiments, abacterium of the invention expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 16 or a polypeptide having the aminoacid sequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 16, wherein in said amino acid sequence at position19 G is replaced by another amino acid. Preferably, the one or moreamino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium which comprises withinthe cycA gene one or more nucleotide substitutions resulting in theamino acid substitution 1220V in the encoded polypeptide. Furtherinformation regarding cycA of, e.g., Escherichia coli is available atEcoCyc (www.biocyc.org) under Accession numbers EG12504. According tocertain embodiments, a bacterium of the invention expresses apolypeptide encoded by the cycA gene, wherein in said polypeptide atposition 220 I is replaced by V. A representative amino acid sequence ofa polypeptide encoded by the cycA gene is set forth in SEQ ID NO: 17.According to particular embodiments, a bacterium of the inventionexpresses a polypeptide having the amino acid sequence set forth in SEQID NO: 17 or a polypeptide having the amino acid sequence which has atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 17,wherein in said amino acid sequence at position at position 220 I isreplaced by V.

According to certain embodiments, the present invention provides abacterium which comprises within the cycA gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the cycA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position 1220. According to particular embodiments, abacterium of the invention expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 17 or a polypeptide having the aminoacid sequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 17, wherein in said amino acid sequence at position220 I is replaced by another amino acid. Preferably, the one or moreamino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium which comprises withinthe rpe gene one or more nucleotide substitutions resulting in the aminoacid substitution I202T in the encoded polypeptide. Further informationregarding rpe of, e.g., Escherichia coli is available at EcoCyc(www.biocyc.org) under Accession numbers M004. According to certainembodiments, a bacterium of the invention expresses a polypeptideencoded by the rpe gene, wherein in said polypeptide at position 202 Iis replaced by T. A representative amino acid sequence of a polypeptideencoded by the rpe gene is set forth in SEQ ID NO: 18. According toparticular embodiments, a bacterium of the invention expresses apolypeptide having the amino acid sequence set forth in SEQ ID NO: 18 ora polypeptide having the amino acid sequence which has at least about90%, at least about 93%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 18, whereinin said amino acid sequence at position at position 202 I is replaced byT.

According to certain embodiments, the present invention provides abacterium which comprises within the rpe gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the rpe gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position 202I. According to particular embodiments, abacterium of the invention expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 18 or a polypeptide having the aminoacid sequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 18, wherein in said amino acid sequence at position202 I is replaced by another amino acid. Preferably, the one or moreamino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium which comprises withinthe yojl gene one or more nucleotide substitutions resulting in theamino acid substitution D334H in the encoded polypeptide. Furtherinformation regarding yojl of, e.g., Escherichia coli is available atEcoCyc (www.biocyc.org) under Accession numbers EG12070. According tocertain embodiments, a bacterium of the invention expresses apolypeptide encoded by the yojl gene, wherein in said polypeptide atposition 334 D is replaced by H. A representative amino acid sequence ofa polypeptide encoded by the yojl gene is set forth in SEQ ID NO: 19.According to particular embodiments, a bacterium of the inventionexpresses a polypeptide having the amino acid sequence set forth in SEQID NO: 19 or a polypeptide having the amino acid sequence which has atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 19,wherein in said amino acid sequence at position at position 334 D isreplaced by H.

According to certain embodiments, the present invention provides abacterium which comprises within the yojl gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the yojl gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position D334. According to particular embodiments, abacterium of the invention expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 19 or a polypeptide having the aminoacid sequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 19, wherein in said amino acid sequence at position334 D is replaced by another amino acid. Preferably, the one or moreamino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium which comprises withinthe hyaF gene one or more nucleotide substitutions resulting in theamino acid substitution V120G in the encoded polypeptide. Furtherinformation regarding hyaF of, e.g., Escherichia coli is available atEcoCyc (www.biocyc.org) under Accession numbers EG10473. According tocertain embodiments, a bacterium of the invention expresses apolypeptide encoded by the hyaF gene, wherein in said polypeptide atposition 120 V is replaced by G. A representative amino acid sequence ofa polypeptide encoded by the hyaF gene is set forth in SEQ ID NO: 20.According to particular embodiments, a bacterium of the inventionexpresses a polypeptide having the amino acid sequence set forth in SEQID NO: 20 or a polypeptide having the amino acid sequence which has atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 20,wherein in said amino acid sequence at position at position 120 V isreplaced by G.

According to certain embodiments, the present invention provides abacterium which comprises within the hyaF gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the hyaF gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position V120. According to particular embodiments, abacterium of the invention expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 20 or a polypeptide having the aminoacid sequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 20, wherein in said amino acid sequence at position120 V is replaced by another amino acid. Preferably, the one or moreamino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium which comprises withinthe pykF gene one or more nucleotide substitutions resulting in theamino acid substitution E250* in the encoded polypeptide, where *designates a stop codon. Alternatively, the pykF gene may comprise oneor more nucleotide substitutions resulting in the termination of theencoded polypeptide at a position upstream of position 250. Furtherinformation regarding pykF of, e.g., Escherichia coli is available atEcoCyc (www.biocyc.org) under Accession numbers EG10804. According tocertain embodiments, a bacterium of the invention expresses apolypeptide encoded by the pykF gene, wherein said polypeptideterminates after position 249 or any position upstream thereof. Arepresentative amino acid sequence of a polypeptide encoded by the pykFgene is set forth in SEQ ID NO: 21. According to particular embodiments,a bacterium of the invention expresses a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 22 or a polypeptide having theamino acid sequence which has at least about 90%, at least about 93%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99%, sequence identity to the amino acid sequenceset forth in SEQ ID NO: 22.

According to other certain embodiments, a bacterium of the invention hasbeen further modified to attenuate the expression of the pykF gene(e.g., by inactivation of the gene). Attenuation, and more particularlyinactivation, of the gene expression can be achieved as described hereinabove. For example, lambda red mediated gene replacement may be used forinactivating gene expression.

The present invention also provides a bacterium which comprises withinthe malT gene one or more nucleotide substitutions resulting in theamino acid substitution Q420* in the encoded polypeptide, where *designates a stop codon. Alternatively, the malT gene may comprise oneor more nucleotide substitutions resulting in the termination of theencoded polypeptide at a position upstream of position 420. Furtherinformation regarding malT of, e.g., Escherichia coli is available atEcoCyc (www.biocyc.org) under Accession numbers EG10562. According tocertain embodiments, a bacterium of the invention expresses apolypeptide encoded by the malT gene, wherein said polypeptideterminates after position 419 or any position upstream thereof. Arepresentative amino acid sequence of a polypeptide encoded by the malTgene is set forth in SEQ ID NO: 23. According to particular embodiments,a bacterium of the invention expresses a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 24 or a polypeptide having theamino acid sequence which has at least about 90%, at least about 93%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99%, sequence identity to the amino acid sequenceset forth in SEQ ID NO: 24.

According to other certain embodiments, a bacterium of the invention hasbeen further modified to attenuate the expression of the malT gene(e.g., by inactivation of the gene). Attenuation, and more particularlyinactivation, of the gene expression can be achieved as described hereinabove. For example, lambda red mediated gene replacement may be used forinactivating gene expression.

The present invention also provides a bacterium which comprises withinthe rpoB gene one or more nucleotide substitutions resulting in theamino acid substitution P520L in the encoded polypeptide. Furtherinformation regarding rpoB of, e.g., Escherichia coli is available atEcoCyc (www.biocyc.org) under Accession numbers EG10894. According tocertain embodiments, a bacterium of the invention expresses apolypeptide encoded by the rpoB gene, wherein in said polypeptide atposition 520 P is replaced by L A representative amino acid sequence ofa polypeptide encoded by the rpoB gene is set forth in SEQ ID NO: 25.According to particular embodiments, a bacterium of the inventionexpresses a polypeptide having the amino acid sequence set forth in SEQID NO: 25 or a polypeptide having the amino acid sequence which has atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 25,wherein in said amino acid sequence at position at position 520 P isreplaced by L.

According to certain embodiments, the present invention provides abacterium which comprises within the rpoB gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the rpoB gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position P520. According to particular embodiments, abacterium of the invention expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 25 or a polypeptide having the aminoacid sequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 25, wherein in said amino acid sequence at position520 P is replaced by another amino acid. Preferably, the one or moreamino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium which comprises withinthe fumB gene one or more nucleotide substitutions resulting in theamino acid substitution T218P in the encoded polypeptide. Furtherinformation regarding fumB of, e.g., Escherichia coli is available atEcoCyc (www.biocyc.org) under Accession numbers EG10357. According tocertain embodiments, a bacterium of the invention expresses apolypeptide encoded by the fumB gene, wherein in said polypeptide atposition 218 T is replaced by P. A representative amino acid sequence ofa polypeptide encoded by the fumB gene is set forth in SEQ ID NO: 26.According to particular embodiments, a bacterium of the inventionexpresses a polypeptide having the amino acid sequence set forth in SEQID NO: 26 or a polypeptide having the amino acid sequence which has atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 26,wherein in said amino acid sequence at position at position 218 T isreplaced by P.

According to certain embodiments, the present invention provides abacterium which comprises within the fumB gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the fumB gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position T218. According to particular embodiments, abacterium of the invention expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 26 or a polypeptide having the aminoacid sequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 26, wherein in said amino acid sequence at position218 T is replaced by another amino acid. Preferably, the one or moreamino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium which comprises withinthe gshA gene one or more nucleotide substitutions resulting in theamino acid substitution A178V in the encoded polypeptide. Furtherinformation regarding gshA of, e.g., Escherichia coli is available atEcoCyc (www.biocyc.org) under Accession numbers EG10418. According tocertain embodiments, a bacterium of the invention expresses apolypeptide encoded by the gshA gene, wherein in said polypeptide atposition 178 A is replaced by V. A representative amino acid sequence ofa polypeptide encoded by the gshA gene is set forth in SEQ ID NO: 27.According to particular embodiments, a bacterium of the inventionexpresses a polypeptide having the amino acid sequence set forth in SEQID NO: 27 or a polypeptide having the amino acid sequence which has atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 27,wherein in said amino acid sequence at position at position 178 A isreplaced by V.

According to certain embodiments, the present invention provides abacterium which comprises within the gshA gene one or more nucleotidesubstitutions resulting in one or more amino acid substitutions whichincrease tolerance towards L-serine. More particularly, a bacterium isprovided which comprises within the gshA gene one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position A178. According to particular embodiments, abacterium of the invention expresses a polypeptide having the amino acidsequence set forth in SEQ ID NO: 27 or a polypeptide having the aminoacid sequence which has at least about 90%, at least about 93%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99%, sequence identity to the amino acid sequence setforth in SEQ ID NO: 27, wherein in said amino acid sequence at position178 A is replaced by another amino acid. Preferably, the one or moreamino acid substitutions are non-conservative substitutions.

The present invention also provides a bacterium which comprises withinthe lamB gene one or more nucleotide substitutions resulting in theamino acid substitution Q112* in the encoded polypeptide, where *designates a stop codon. Alternatively, the lamB gene may comprise oneor more nucleotide substitutions resulting in the termination of theencoded polypeptide at a position upstream of position 112. Furtherinformation regarding lamB of, e.g., Escherichia coli such as nucleotidesequence of the gene or amino acid sequence of the encoded polypeptideis available at EcoCyc (www.biocyc.org) under Accession numbers EG10528.According to certain embodiments, a bacterium of the invention expressesa polypeptide encoded by the lamB gene, wherein said polypeptideterminates after position 111 or any position upstream thereof. Arepresentative amino acid sequence of a polypeptide encoded by the lamBgene is set forth in SEQ ID NO: 28. According to particular embodiments,a bacterium of the invention expresses a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 29 or a polypeptide having theamino acid sequence which has at least about 90%, at least about 93%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99%, sequence identity to the amino acid sequenceset forth in SEQ ID NO: 29.

According to other certain embodiments, a bacterium of the invention hasbeen further modified to attenuate the expression of the lamB gene(e.g., by inactivation of the gene). Attenuation, and more particularlyinactivation, of the gene expression can be achieved as described hereinabove. For example, lambda red mediated gene replacement may be used forinactivating gene expression.

A bacterium of the present invention may comprise one or more, such astwo or more, three or more, four or more, or five or more, genemutations as mentioned.

For example, the bacterium may comprise one or more (such as two ormore) gene mutations selected from the group consisting of: one or morenucleotide substitutions within the Irp gene resulting in an amino acidsubstitution at position D143 in the encoded polypeptide, one or morenucleotide substitutions within the rho gene resulting in an amino acidsubstitution at position R87 in the encoded polypeptide, one or morenucleotide substitutions within the eno gene resulting in an amino acidsubstitution at position V164 in the encoded polypeptide, and one ormore nucleotide substitutions within the argP gene resulting in an aminoacid substitution at position V164 in the encoded polypeptide.

For example, the bacterium may comprise one or more (such as two ormore) gene mutations selected from the group consisting of: one or morenucleotide substitutions within the Irp gene resulting in the amino acidsubstitution D143G in the encoded polypeptide, one or more nucleotidesubstitutions within the rho gene resulting in the amino acidsubstitution R87L in the encoded polypeptide, one or more nucleotidesubstitutions within the eno gene resulting in the amino acidsubstitution V164L in the encoded polypeptide, and one or morenucleotide substitutions within the argP gene resulting in the aminoacid substitution V164L in the encoded polypeptide.

According to certain embodiments, a bacterium of the invention compriseswithin the thrA gene one or more nucleotide substitutions resulting inan amino acid substitution in the encoded polypeptide at position Y356,said substitution being selected from the group consisting of Y356C,Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F,Y356A, Y356I, Y356P, Y356H, Y356R and Y356L; one or more nucleotidesubstitutions resulting in an amino add substitution in the encodedpolypeptide at position S357, said substitution being selected from thegroup consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A,S357N, S357F, S357H, S357K, S357I and S357M; and/or one or morenucleotide substitutions resulting in an amino add substitution in theencoded polypeptide at position S359, said substitution being selectedfrom the group consisting of S359R, S359G, S359M, S359F, S359T, S359P,S359V, S359Q. S359A, S359C, S359K, S359E and S359L; and one or more(such as two or more) gene mutations selected from the group consistingof: one or more nucleotide substitutions within the Irp gene resultingin an amino acid substitution at position D143 (such as D143G) in theencoded polypeptide, one or more nucleotide substitutions within the rhogene resulting in the amino acid substitution at position R87 (such asR87L) in the encoded polypeptide, one or more nucleotide substitutionswithin the eno gene resulting in the amino acid substitution at positionV164 (such as V164L) in the encoded polypeptide, and one or morenucleotide substitutions within the argP gene resulting in an amino acidsubstitution at position V164 (such as V164L) in the encodedpolypeptide.

According to certain embodiments, a bacterium of the present inventioncomprises one or more nucleotide substitutions within the thrA generesulting in one or more amino acid substitution in the encodedpolypeptide selected from the group consisting of Y356C, S357R andS359R; and one or more (such as two or more) gene mutations selectedfrom the group consisting of: one or more nucleotide substitutionswithin the Irp gene resulting in the amino acid substitution D143G inthe encoded polypeptide, one or more nucleotide substitutions within therho gene resulting in the amino acid substitution R87L In the encodedpolypeptide, one or more nucleotide substitutions within the eno generesulting in the amino acid substitution V164L in the encodedpolypeptide, and one or more nucleotide substitutions within the argPgene resulting in the amino acid substitution V164L in the encodedpolypeptide.

According to certain embodiments, a bacterium of the present inventioncomprises one or more nucleotide substitutions within the thrA generesulting in the amino acid substitution Y356C in the encodedpolypeptide; and one or more (such as two or more) gene mutationsselected from the group consisting of: one or more nucleotidesubstitutions within the Irp gene resulting in the amino acidsubstitution D143G in the encoded polypeptide, one or more nucleotidesubstitutions within the rho gene resulting in the amino acidsubstitution R87L in the encoded polypeptide, one or more nucleotidesubstitutions within the eno gene resulting in the amino acidsubstitution V164L in the encoded polypeptide, and one or morenucleotide substitutions within the argP gene resulting in the aminoacid substitution V164L in the encoded polypeptide.

According to certain embodiments, a bacterium of the present inventioncomprises one or more nucleotide substitutions within the thrA generesulting in the amino acid substitution S357R in the encodedpolypeptide; and one or more (such as two or more) gene mutationsselected from the group consisting of: one or more nucleotidesubstitutions within the Irp gene resulting in the amino acidsubstitution D143G in the encoded polypeptide, one or more nucleotidesubstitutions within the rho gene resulting in the amino acidsubstitution R87L in the encoded polypeptide, one or more nucleotidesubstitutions within the eno gene resulting in the amino acidsubstitution V164L in the encoded polypeptide, and one or morenucleotide substitutions within the argP gene resulting in the aminoacid substitution V164L in the encoded polypeptide.

According to certain embodiments, a bacterium of the present inventioncomprises one or more nucleotide substitutions within the thrA generesulting in the substitution S359R in the encoded polypeptide; and oneor more (such as two or more) gene mutations selected from the groupconsisting of: one or more nucleotide substitutions within the Irp generesulting in the amino add substitution D143G in the encodedpolypeptide, one or more nucleotide substitutions within the rho generesulting in the amino acid substitution R87L in the encodedpolypeptide, one or more nucleotide substitutions within the eno generesulting in the amino acid substitution V164L in the encodedpolypeptide, and one or more nucleotide substitutions within the argPgene resulting in the amino acid substitution V164L in the encodedpolypeptide.

According to certain embodiments, a bacterium of the invention compriseswithin its genome a deletion of the first 5 bp of gene rhtA gene, acomplete deletion of genes ompX and ybiP, a deletion of 239 bp of sRNArybA and a deletion of 77 bp of gene mnt5. According to certainembodiments, the bacterium comprises within its genome a deletion ofabout 2854 bp from a location which corresponds to location 850092 inthe genome sequence NC_000913. This deletion results in a deletion ofthe first 5 bp of gene rhtA gene, a complete deletion of genes ompX andybiP, a deletion of 239 bp of sRNA rybA and a 77 bp deletion of genemnt5. Such deletion can be achieved by using the lambda-red orcam-sacB-system.

According to certain embodiments, a bacterium of the invention compriseswithin its genome an insertion of an insertion sequence element IS1(e.g., having a length of about 768 bp) in the intergenic region betweengenes trxA and rho. According to certain embodiments, the bacteriumcomprises within its genome an insertion of an insertion sequenceelement IS1 (e.g., having a length of about 768 bp) in the laggingstrand at a location which corresponds to location 3966174 in the genomesequence NC_000913. According to particular embodiments, the bacteriumfurther comprises a duplication of around 9 bp upstream and downstreamof insertion sequence.

According to certain embodiments, a bacterium of the invention compriseswithin its genome an insertion of 1 bp in the intergenic region betweengenes gcvA and ygdI. According to certain embodiments, the bacteriumcomprises within its genome an insertion of 1 bp at a location whichcorresponds to location 2942629 in the genome sequence NC_000913.

According to certain embodiments, a bacterium of the invention compriseswithin its genome an insertion of an insertion sequence element 154(e.g., having a length of about 1342 bp) in the intergenic regionbetween genes gcvA and ygdI. According to certain embodiments, thebacterium comprises within its genome an insertion of an insertionsequence element 15S4 (e.g., having a length of about 1342 bp) at alocation which corresponds to location 2942878 in the genome sequenceNC_000913. According to particular embodiments, the bacterium furthercomprises a duplication of about 13 bp upstream and downstream ofinsertion sequence.

According to certain embodiments, a bacterium of the invention compriseswithin its genome an insertion of 1 bp in the intergenic region betweengenes dopA and gcvR. According to certain embodiments, the bacteriumcomprises within its genome an insertion of 1 bp at a location whichcorresponds to location 2599854 in the genome sequence NC_000913.

According to certain embodiments, a bacterium of the invention compriseswithin its genome an insertion of an insertion sequence element IS1(e.g., having a length of about 768 bp) which lead to a truncation ofgene frc. According to certain embodiments, the bacterium compriseswithin its genome an insertion sequence element IS1 (e.g., having alength of about 768 bp) in the lagging strand at a location whichcorresponds to location 2492323 in the genome sequence NC_000913.According to particular embodiments, the bacterium further comprises aduplication of 9 bp upstream and downstream of insertion sequence.

According to other certain embodiments, a bacterium of the invention hasbeen further modified to attenuate the expression of the frc gene (e.g.,by inactivation of the gene). Attenuation, and more particularlyinactivation, of the gene expression can be achieved as described hereinabove. For example, lambda red mediated gene replacement may be used forinactivating gene expression.

According to certain embodiments, a bacterium of the invention compriseswithin its genome an insertion of an insertion sequence element IS55(e.g., having a length of about 1195 bp) which leads to deletion of themajority of gene aroP. According to certain embodiments, the bacteriumcomprises within its genome an insertion sequence element IS5 (e.g.,having a length of about 1195 bp) at a location which corresponds tolocation 121518 in the genome sequence NC_000913. According toparticular embodiments, the bacterium further comprises a duplication of4 bp upstream and downstream of insertion sequence.

According to other certain embodiments, a bacterium of the invention hasbeen further modified to attenuate the expression of the aroP gene(e.g., by inactivation of the gene). Attenuation, and more particularlyinactivation, of the gene expression can be achieved as described hereinabove. For example, lambda red mediated gene replacement may be used forinactivating gene expression.

According to certain embodiments, a bacterium of the invention compriseswithin its genome an insertion of an insertion sequence element IS51(e.g., having a length of about 768 bp) in the intergenic region betweengenes mdtJ and tqsA. According to certain embodiments, the bacteriumcomprises within its genome an insertion sequence element IS1 (e.g.,having a length of about 768 bp) in the lagging strand at a locationwhich corresponds to location 1673670 in the genome sequence NC_000913.According to particular embodiments, the bacterium further comprises aduplication of around 9 bp upstream and downstream of insertionsequence.

According to certain embodiments, a bacterium of the invention compriseswithin its genome a nucleotide substitution, such as a C→T substitution,within the intergenic region between genes trxB and Irp. According tocertain embodiments, the bacterium comprises within its genome anucleotide substitution, such as a C→T substitution, at a location whichcorresponds to location 923321 in the genome sequence NC_000913. Suchmutation is 271 bp upstream of trxB and 274 bp upstream of Irp.

According to certain embodiments, a bacterium of the invention compriseswithin its genome a nucleotide substitution, such as a T→C substitution,within the intergenic region between genes yftB and fklB. According tocertain embodiments, the bacterium comprises within its genome anucleotide substitution, such as a T→C substitution, at a location whichcorresponds to location 4428871 in the genome sequence NC_000913. Suchmutation is 154 bp upstream of of yftB and 64 bp upstream of fklB.

As further demonstrate herein, attenuating (e.g., by inactivating of thegene) the expression of a gene coding for a polypeptide having Glucose6-phosphate-1-dehydrogenase (G6PDH) activity in a reversed engineeredstrain resulted in a significantly increased production and yield ofL-serine from glucose as shown in Table 59 (Example 8).

Therefore, according to certain embodiments, a bacterium of theinvention has been modified to attenuate expression of a gene coding fora polypeptide having glucose 6-phosphate-1-dehydrogenase (G6PDH)activity. More particularly, the present invention provides a bacteriumwhich has been modified to attenuate the expression of the gene zwf.Further information regarding zwf of, e.g., Escherichia coli isavailable at EcoCyc (www.biocyc.org) under Accession numbers EG11221. Arepresentative nucleotide sequence of zwf is set forth in SEQ ID NO: 30.

The gene expression may be attenuated by inactivation of the gene. Thus,a bacterium according to the invention can be one which has beenmodified to inactivate the gene coding for a polypeptide having Glucose6-phosphate-1-dehydrogenase (G6PDH) activity (e.g, by inactivation ofthe gene).

Attenuation, and more particularly inactivation, of the gene expressioncan be achieved as described herein above. For example, lambda redmediated gene replacement may be used for inactivating gene expression.

According to certain embodiments, a bacterium of the invention expressesa polypeptide encoded by the brnQ gene, wherein said polypeptideterminates after position 308 or any position upstream thereof.According to other certain embodiments, a bacterium of the invention hasbeen further modified to attenuate the expression of the brnQ gene(e.g., by inactivation of the gene). Attenuation, and more particularlyinactivation, of the gene expression can be achieved as described hereinabove. For example, lambda red mediated gene replacement may be used forinactivating gene expression.

According to certain embodiments, a bacterium of the invention has beenfurther modified to attenuate expression of a gene coding for apolypeptide having glucose 6-phosphate-1-dehydrogenase (G6PDH) activity;express a polypeptide encoded by the thrA gene, wherein in saidpolypeptide at position 357 serine is replaced by arginine; expresses apolypeptide encoded by the rho gene, wherein in said polypeptide atposition 87 R is replaced by L; and expresses a polypeptide encoded bythe brnQ gene, wherein said polypeptide terminates after position 308 orany position upstream thereof. Alternatively, the bacterium may bemodified to attenuate the expression of the brnQ gene (e.g., byinactivation of the gene). Suitable methods for attenuation of geneexpression are described above.

Further information regarding brnQ of, e.g., Escherichia coli isavailable at EcoCyc (www.biocyc.org) under Accession numbers EG12168. Arepresentative amino acid sequence of brnmQ is set forth in SEQ ID NO:31.

According to particular embodiments, the bacterium has been furthermodified to attenuate expression of the gene zwf (e.g, by inactivationof the gene); expresses a polypeptide having the amino acid sequence setforth in SEQ ID NO: 11 or a polypeptide having at least about 90%, atleast about 93%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, sequence identity to theamino acid sequence set forth in SEQ ID NO: 11, wherein in said aminoacid sequence at position 357 serine is replaced by arginine; express apolypeptide having the amino acid sequence set forth in SEQ ID NO: 13 ora polypeptide having the amino acid sequence which has at least about90%, at least about 93%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99%, sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 13, whereinin said amino acid sequence at position 87 R is replaced by L; andexpresses a polypeptide having the amino acid sequence set forth in SEQID NO: 32 or a polypeptide having the amino add sequence which has atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 32.

According to particular embodiments, the bacterium has been furthermodified to attenuate expression of the gene zwf and brnQ (e.g, byinactivation of the genes); and expresses a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 11 or a polypeptide having atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%,sequence identity to the amino acid sequence set forth in SEQ ID NO: 11,wherein in said amino acid sequence at position 357 serine is replacedby arginine; express a polypeptide having the amino acid sequence setforth in SEQ ID NO: 13 or a polypeptide having the amino acid sequencewhich has at least about 90%, at least about 93%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99%, sequence identity to the amino acid sequence set forth in SEQID NO: 13, wherein in said amino acid sequence at position 87 R isreplaced by L.

As detailed above, a bacterium of the invention may have been modifiedto overexpress certain polypeptides as detailed herein, which means thatan exogenous nucleic acid molecule, such as a DNA molecule, whichcomprises a nucleotide sequence encoding said polypeptide has beenintroduced in the bacterium. Techniques for introducing exogenousnucleic acid molecule, such as a DNA molecule, into a bacterial cellsare well-known to those of skill in the art, and include transformation(e.g., heat shock or natural transformation) among others.

In order to facilitate overexpression of a polypeptide in the bacterium,the exogenous nucleic acid molecule may comprise suitable regulatoryelements such as a promoter that is functional in the bacterial cell tocause the production of an mRNA molecule and that is operably linked tothe nucleotide sequence encoding said polypeptide.

Promoters useful in accordance with the invention are any knownpromoters that are functional in a given host cell to cause theproduction of an mRNA molecule. Many such promoters are known to theskilled person. Such promoters include promoters normally associatedwith other genes, and/or promoters isolated from any bacteria. The useof promoters for protein expression is generally known to those ofskilled in the art of molecular biology, for example, see Sambrook etal., Molecular cloning: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989. The promoter employed may beinducible, such as a temperature inducible promoter (e.g., a pL or pRphage lambda promoters, each of which can be controlled by thetemperature-sensitive lambda repressor c1857). The term “inducible” usedin the context of a promoter means that the promoter only directstranscription of an operably linked nucleotide sequence if a stimulus ispresent, such as a change in temperature or the presence of a chemicalsubstance (“chemical inducer”). As used herein, “chemical induction”according to the present invention refers to the physical application ofan exogenous or endogenous substance (incl. macromolecules, e.g.,proteins or nucleic acids) to a host cell. This has the effect ofcausing the target promoter present in the host cell to increase therate of transcription. Alternatively, the promoter employed may beconstitutive. The term “constitutive” used in the context of a promotermeans that the promoter is capable of directing transcription of anoperably linked nucleotide sequence in the absence of stimulus (such asheat shock, chemicals etc.).

Temperature induction systems work, for example, by employing promotersthat are repressed by thermolabile repressors. These repressors areactive at lower temperatures for example at 30° C., while unable to foldcorrectly at 37° C. and are therefore inactive. Such circuits thereforecan be used to directly regulate the genes of interest (St-Pierre et al.2013) also by genome integration of the genes along with the repressors.Examples of such as a temperature inducible expression system are basedon the pL and/or pR A phage promoters which are regulated by thethermolabile c1857 repressor. Similar to the genome integrated DE3system, the expression of the T7 RNA polymerase gene may also becontrolled using a temperature controlled promoter system (Mertens etal. 1995), while the expression of the genes of interest can becontrolled using a T7 promoter.

Non-limiting examples of promoters functional in bacteria, such asEscherichia coli, include both constitutive and inducible promoters suchas T7 promoter, the beta-lactamase and lactose promoter systems;alkaline phosphatase (phoA) promoter, a tryptophan (trp) promotersystem, tetracycline promoter, lambda-phage promoter, ribosomal proteinpromoters; and hybrid promoters such as the tac promoter. Otherbacterial and synthetic promoters are also suitable.

Besides a promoter, the exogenous nucleic acid molecule may furthercomprise at least one regulatory element selected from a 5′ untranslatedregion (5′UTR) and 3′ untranslated region (3′ UTR). Many such 5′ UTRsand 3′ UTRs derived from prokaryotes and eukaryotes are well known tothe skilled person. Such regulatory elements include 5′ UTRs and 3′ UTRsnormally associated with other genes, and/or 5′ UTRs and 3′ UTRsisolated from any bacteria.

Usually, the 5′ UTR contains a ribosome binding site (RBS), also knownas the Shine Dalgarno sequence which is usually 3-10 base pairs upstreamfrom the initiation codon.

The exogenous nucleic acid molecule may be a vector or part of a vector,such as an expression vector. Normally, such a vector remainsextrachromosomal within the bacterial cell which means that it is foundoutside of the nucleus or nucleoid region of the bacterium.

It is also contemplated by the present invention that the exogenousnucleic acid molecule is stably integrated into the genome of thebacterium. Means for stable integration into the genome of a host cell,e.g., by homologous recombination, are well known to the skilled person.

A bacterium in accordance with the present invention can be producedfrom any suitable bacterium, such as a Gram-positive or Gram-negativebacterium.

Examples of bacteria which can be used to derive a bacterium of theinvention belong to the Enterobacteriaceae family, such as bacteriabelonging to a genus selected from the group consisting of Escherichia,Arsenophonus, Biostraticola, Brenneria, Buchnera, Budvicia,Buttiauxella, Cedecea, Citrobacter, Cosenzaea, Cronobacter, Dickeya,Edwardsiella, Enterobacter, Erwinia, Ewingella, Gibbsiella, Hofnia,Klebsiella, Leclercia, Leminorella, Lonsdalea, Mangrovibacter,Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium,Phaseolibacter, Photorhabdus, Plesiomonas, Proteus, Rahnella,Raoultella, Sacchorobacter, Salmonella, Samsonia, Serratia, Shimwellia,Sodolis, Tatumella, Thorsellia, Trobulsiella, Wigglesworthia, Yersiniaand Yokenella.

According to certain other embodiments, the bacterium belongs to a genusselected from the group selected from Escherichia, Bacillus,Loctococcus, Lactobacillus, Clostridium, Corynebacterium, Geobacillus,Streptococcus, Pseudomonos, Streptomyces, Shigella, Acinetobacter,Citrobacter, Salmonella, Klebsiella, Enterobacter, Erwinia, Kluyvera,Serratia, Cedecea, Morganella, Hofnia, Edwardsiella, Providencia,Proteus and Yersinia.

According to particular embodiments, the bacterium belongs to the genusEscherichia. According to particular embodiments, the bacterium isEscherichia coli. Non-limiting examples of a bacterium belonging to thegenus Escherichia, which can be used to derive a bacterium of theinvention, are Escherichia coli K-12 (especially substrain MG1655 orW3110), B121, W, or Crooks. According to more particular embodiments,the bacterium is Escherichia coli K-12.

According to other particular embodiments, the bacterium belongs to thegenus Corynebacterium. A non-limiting example of a bacterium of thegenus Corynebacterium is Corynebacterium glutamicum. According to moreparticular embodiments, the bacterium is Corynebacterium glutamicum.

According to other particular embodiments, the bacterium belongs to thegenus Bacillus. Non-limiting examples of a bacterium of the genusBacillus are Bacillus subtitlis, Bacillus amyloliquefaciens, Bacilluslicheniformis, and Bacillus mojavensis. According to more particularembodiments, the bacterium is Bacillus subtitlis. According to othermore particular embodiments, the bacterium is Bacillus licheniformis.

According to other particular embodiments, the bacterium belongs to thegenus Loctococcus. A non-limiting example of a bacterium of the genusLactococcus is Lactococcus lactis. According to more particularembodiments, the bacterium is Loctococcus lactis.

According to other particular embodiments, the bacterium belongs to thegenus Streptomyces. A non-limiting examples of a bacterium of the genusStreptomyces are Streptomyces lividans, Streptomyces coelicolar, orStreptomyces griseus. According to more particular embodiments, thebacterium is Streptomyces lividans. According to other more particularembodiments, the bacterium is Streptomyces coelicolar. According toother more particular embodiments, the bacterium is Streptomycesgriseus.

According to other particular embodiments, the bacterium belongs to thegenus Pseudomonas. A non-limiting example of a bacterium of the genusPseudomonos is Pseudomonos putida. According to more particularembodiments, the bacterium is Pseudomonas putida.

Method of the Invention

The present invention also provides methods for producing L-serine or aL-serine derivative using a bacterium according to the presentinvention. Particularly, the present invention provides a method forproducing L-serine or a L-serine derivative, said method comprisescultivating a bacterium as detailed herein in a culture medium.

According to certain embodiments, present invention provides a methodfor producing L-serine. Particularly, the present invention provides amethod for producing L-serine, said method comprises cultivating abacterium as detailed herein in a culture medium. The method may furthercomprise collecting L-serine from the culture medium.

According to certain embodiments, present invention provides a methodfor producing a L-serine derivative. Particularly, the present inventionprovides a method for producing L-serine derivative, said methodcomprises cultivating a bacterium as detailed herein in a culturemedium. The L-serine derivative may be selected from the groupconsisting of L-cysteine, L-methionine, L-glycine, O-acetylserine,L-tryptophan, thiamine, ethanolamine and ethylene glycol. The method mayfurther comprise collecting the L-serine derivative from the culturemedium.

According to certain embodiments, present invention provides a methodfor producing L-cysteine. Particularly, the present invention provides amethod for producing L-cysteine, said method comprises cultivating abacterium as detailed herein in a culture medium. The method may furthercomprise collecting L-cysteine from the culture medium.

According to certain embodiments, present invention provides a methodfor producing L-methionine. Particularly, the present invention providesa method for producing L-methionine; said method comprises cultivating abacterium as detailed herein in a culture medium. The method may furthercomprise collecting L-methionine from the culture medium.

According to certain embodiments, present invention provides a methodfor producing L-glycine. Particularly, the present invention provides amethod for producing L-glycine; said method comprises cultivating abacterium as detailed herein in a culture medium. The method may furthercomprise collecting L-glycine from the culture medium.

According to certain embodiments, present invention provides a methodfor producing O-acetylserine. Particularly, the present inventionprovides a method for producing O-acetylserine; said method comprisescultivating a bacterium as detailed herein in a culture medium. Themethod may further comprise collecting O-acetylserine from the culturemedium.

According to certain embodiments, present invention provides a methodfor producing L-tryptophan. Particularly, the present invention providesa method for producing L-tryptophan; said method comprises cultivating abacterium as detailed herein in a culture medium. The method may furthercomprise collecting L-tryptophan from the culture medium.

According to certain embodiments, present invention provides a methodfor producing thiamine. Particularly, the present invention provides amethod for producing thiamine; said method comprises cultivating abacterium as detailed herein in a culture medium. The method may furthercomprise collecting thiamine from the culture medium.

According to certain embodiments, present invention provides a methodfor producing ethanolamine. Particularly, the present invention providesa method for producing ethanolamine; said method comprises cultivating abacterium as detailed herein in a culture medium. The method may furthercomprise collecting ethanolamine from the culture medium.

According to certain embodiments, present invention provides a methodfor producing ethylene glycol. Particularly, the present inventionprovides a method for producing ethylene glycol; said method comprisescultivating a bacterium as detailed herein in a culture medium. Themethod may further comprise collecting ethylene glycol from the culturemedium.

The culture medium employed may be any conventional medium suitable forculturing a bacterium cell in question, and may be composed according tothe principles of the prior art. The medium will usually contain allnutrients necessary for the growth and survival of the respectivebacterium, such as carbon and nitrogen sources and other inorganicsalts. Suitable media, e.g. minimal or complex media, are available fromcommercial suppliers, or may be prepared according to publishedreceipts, e.g. the American Type Culture Collection (ATCC) Catalogue ofstrains. Non-limiting standard medium well known to the skilled personinclude Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, MSbroth, Yeast Peptone Dextrose, BMMY, GMMY, or Yeast Malt Extract (YM)broth, which are all commercially available. A non-limiting example ofsuitable media for culturing bacterial cells, such as E. coli cells,including minimal media and rich media such as Luria Broth (LB), M9media, M17 media, SA media, MOPS media, Terrific Broth, YT and others.

In order to further increase the yield of L-serine or L-serinederivative, the culture medium may further be supplemented withL-threonine. The culture medium may generally contain L-threonine at aconcentration from about 0.05 to about 10 g/L, such as from about 0.05to about 5 g/L, from about 0.05 to about 2.5 g/L, from about 0.05 toabout 1 g/L or from about 0.05 to about 0.5 g/L. According to certainembodiments, the culture medium contains L-threonine at a concentrationfrom about 0.05 to about 5 g/L According to certain other embodiments,the culture medium contains L-threonine at a concentration from about0.05 to about 2.5 g/L. According to certain other embodiments, theculture medium contains L-threonine at a concentration from about 0.05to about 1 g/L According to certain other embodiments, the culturemedium contains L-threonine at a concentration from about 0.05 to about0.5 g/L. According to certain other embodiments, the culture mediumcontains L-threonine at a concentration from about 0.1 to about 1 g/LAccording to other embodiments, the culture medium contains L-threonineat a concentration from about 0.2 to about 1 g/L

The carbon source may be any suitable carbon substrate known in the art,and in particularly any carbon substrate commonly used in thecultivation of bacteria and/or fermentation. Non-limiting examples ofsuitable fermentable carbon substrates are C5 sugars (such as arabinoseor xylose), C6 sugars (such as glucose), acetate, glycerol, plant oils,yeast extract, peptone, casaminoacids or mixtures thereof. A carbonsource of particular interest is a C6 sugar such as glucose.

As the nitrogen source, various ammonium salts such as ammonia andammonium sulfate, other nitrogen compounds such as amines, a naturalnitrogen source such as peptone, soybean-hydrolysate, and digestedfermentative microorganism can be used. As minerals, potassiummonophosphate, magnesium sulfate, sodium chloride, ferrous sulfate,manganese sulfate, calcium chloride, and the like can be used.

The cultivation can be preferably performed under aerobic conditions,such as by a shaking culture, and by a stirring culture with aeration,at a temperature of about 20 to about 40° C., such as about 30 to 38°C., preferably about 37° C. The pH of the culture is usually from about5 and about 9, such as from about 6.5 and 7.5. The pH of the culture canbe adjusted with ammonia, calcium carbonate, various acids, variousbases, and buffers. Usually, 1 to 5-day cultivation leads toaccumulation of L-serine in the culture medium.

After cultivation, solids such as cells can be removed from the culturemedium by centrifugation or membrane filtration. L-serine or theL-serine derivative can be collected by conventional method forisolation and purification chemical compounds from a medium. Well-knownpurification procedures include, but are not limited to, centrifugationor filtration, precipitation, ion exchange, chromatographic methods suchas e.g. ion exchange chromatography or gel filtration chromatography,and crystallization methods.

The present invention thus provides L-serine or a L-serine derivativeobtainable by a method as detailed herein.

Certain Other Definitions

The term “bacterium having ability to produce L-serine” as used hereinmeans a bacterium which is able to produce and cause accumulation ofL-serine in a culture medium, can mean that the bacterium is able tocause accumulation in an amount not less than 0.4 g/L, when cultured inminimal M9 media supplemented with 2 g/L glucose, 2 mM glycine and 1 mMthreonine at 37′C with adequate aeration for 40 hours.

The phrase “bacterium which has been modified to attenuate expression ofgenes encoding polypeptides having serine deaminase activity” as usedherein means that the bacterium has been modified in such a way that themodified bacterium contains a reduced amount of the polypeptides havingserine deaminase activity. More particularly, the phrase means that thebacterium is unable to synthesize polypeptides having serine deaminaseactivity.

The phrase “bacterium which has been modified to attenuate expression ofthe gene encoding a polypeptide having serine hydroxymethyltransferaseactivity” as used herein means that the bacterium has been modified insuch a way that the modified bacterium contains a reduced amount of thepolypeptide having serine hydroxymethyltransferase activity. Moreparticularly, the phrase means that the bacterium is unable tosynthesize a polypeptide having serine hydroxymethyltransferaseactivity.

The phrase “bacterium which has been modified to attenuate expression ofthe gene encoding a polypeptide having glucose6-phosphate-1-dehydrogenase (G6PDH) activity” as used herein means thatthe bacterium has been modified in such a way that the modifiedbacterium contains a reduced amount of the polypeptide having glucose6-phosphate-1-dehydrogenase (G6PDH) activity. More particularly, thephrase means that the bacterium is unable to synthesize a polypeptidehaving glucose 6-phosphate-1-dehydrogenase (G6PDH) activity.

The phrase “yeast which has been modified to attenuate expression of agene encoding a polypeptide having serine deaminase activity” as usedherein means that the yeast has been modified in such a way that themodified yeast contains a reduced amount of the polypeptide havingserine deaminase activity. More particularly, the phrase means that theyeast is unable to synthesize a polypeptide having serine deaminaseactivity.

The phrase “yeast which has been modified to attenuate expression ofgenes encoding polypeptides having serine hydroxymethyltransferaseactivity” as used herein means that the yeast has been modified in sucha way that the modified yeast contains a reduced amount of thepolypeptides having serine hydroxymethyltransferase activity. Moreparticularly, the phrase means that the yeast is unable to synthesizepolypeptides having serine hydroxymethyltransferase activity.

The phrase “inactivation of a gene” can mean that the modified geneencodes a completely non-functional protein. It is also possible thatthe modified DNA region is unable to naturally express the gene due tothe deletion of a part of or the entire gene sequence, the shifting ofthe reading frame of the gene, the introduction of missense/nonsensemutation(s), or the modification of an adjacent region of the gene,including sequences controlling gene expression, such as a promoter,enhancer, attenuator, ribosome-binding site, etc. Preferably, a gene ofinterest is inactivated by deletion of a part of or the entire genesequence, such as by gene replacement.

The presence or absence of a gene on the chromosome of a bacterium canbe detected by well-known methods, including PCR, Southern blotting, andthe like. In addition, the level of gene expression can be estimated bymeasuring the amount of mRNA transcribed from the gene using variouswell-known methods, including Northern blotting, quantitative RT-PCR,and the like. The amount of the protein encoded by the gene can bemeasured by well-known methods, including SDS-PAGE followed by animmunoblotting assay (Western blotting analysis), and the like.

“Polypeptide” and “protein” are used interchangeably herein to denote apolymer of at least two amino acids covalently linked by an amide bond,regardless of length or post-translational modification (e.g.,glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Nucleic acid” or “polynucleotide” are used interchangeably herein todenote a polymer of at least two nucleic acid monomer units or bases(e.g., adenine, cytosine, guanine, thymine) covalently linked by aphosphodiester bond, regardless of length or base modification.

“Recombinant” or “non-naturally occurring” when used with reference to,e.g., a host cell, nucleic acid, or polypeptide, refers to a material,or a material corresponding to the natural or native form of thematerial, that has been modified in a manner that would not otherwiseexist in nature, or is identical thereto but produced or derived fromsynthetic materials and/or by manipulation using recombinant techniques.Non-limiting examples include, among others, recombinant host cellsexpressing genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise expressed ata different level.

“Heterologous” as used herein means that a polypeptide is normally notfound in or made (i.e. expressed) by the host organism, but derived froma different species.

“Substitution” or “substituted” refers to modification of thepolypeptide by replacing one amino acid residue with another, forinstance the replacement of an Serine residue with a Glycine or Alanineresidue in a polypeptide sequence is an amino acid substitution. Whenused with reference to a polynucleotide, “substitution” or “substituted”refers to modification of the polynucleotide by replacing one nucleotidewith another, for instance the replacement of a cytosine with a thyminein a polynucleotide sequence is a nucleotide substitution.

“Conservative substitution”, when used with reference to a polypeptide,refers to a substitution of an amino acid residue with a differentresidue having a similar side chain, and thus typically involvessubstitution of the amino acid in the polypeptide with amino acidswithin the same or similar class of amino acids. By way of example andnot limitation, an amino acid with an aliphatic side chain may besubstituted with another aliphatic amino acid, e.g., alanine, valine,leucine, and isoleucine; an amino acid with hydroxyl side chain issubstituted with another amino acid with a hydroxyl side chain, e.g.,serine and threonine; an amino acid having an aromatic side chain issubstituted with another amino acid having an aromatic side chain, e.g.,phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with abasic side chain is substituted with another amino add with a basic sidechain, e.g., lysine and arginine; an amino acid with an acidic sidechain is substituted with another amino acid with an acidic side chain,e.g., aspartic add or glutamic add; and a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively.

“Non-conservative substitution”, when used with reference to apolypeptide, refers to a substitution of an amino acid in a polypeptidewith an amino acid with significantly differing side chain properties.Non-conservative substitutions may use amino acids between, rather thanwithin, the defined groups and affects (a) the structure of the peptidebackbone in the area of the substitution (e.g., serine for glycine), (b)the charge or hydrophobicity, or (c) the bulk of the side chain. By wayof example and not limitation, an exemplary non-conservativesubstitution can be an acidic amino acid substituted with a basic oraliphatic amino acid; an aromatic amino aid substituted with a smallamino acid; and a hydrophilic amino acid substituted with a hydrophobicamino acid.

“Deletion” or “deleted”, when used with reference to a polypeptide,refers to modification of the polypeptide by removal of one or moreamino acids in the reference polypeptide. Deletions can comprise removalof 1 or more amino acids, 2 or more amino adds, 5 or more amino acids,10 or more amino acids, 15 or more amino acids, or 20 or more aminoacids, up to 10% of the total number of amino acids, or up to 20% of thetotal number of amino acids making up the polypeptide while retainingenzymatic activity and/or retaining the improved properties of anengineered enzyme. Deletions can be directed to the internal portionsand/or terminal portions of the polypeptide, in various embodiments, thedeletion can comprise a continuous segment or can be discontinuous.

“Insertion” or “inserted”, when used with reference to a polypeptide,refers to modification of the polypeptide by addition of one or moreamino acids to the reference polypeptide. Insertions can compriseaddition of 1 or more amino acids, 2 or more amino adds, 5 or more aminoacids, 10 or more amino acids, 15 or more amino acids, or 20 or moreamino acids. Insertions can be in the internal portions of thepolypeptide, or to the carboxy or amino terminus. The insertion can be acontiguous segment of amino acids or separated by one or more of theamino acids in the reference polypeptide.

“Expression” includes any step involved in the production of apolypeptide (e.g., encoded enzyme) including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

As used herein, “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid molecule to which it has been linked.One type of vector is a “plasmid”, which refers to a circular doublestranded nucleic acid loop into which additional nucleic acid segmentscan be ligated. Certain vectors are capable of directing the expressionof genes to which they are operatively linked. Such vectors are referredto herein as “expression vectors”. Certain other vectors are capable offacilitating the insertion of an exogenous nucleic acid molecule into agenome of a bacterium. Such vectors are referred to herein as“transformation vectors”. In general, vectors of utility in recombinantnucleic acid techniques are often in the form of plasmids. In thepresent specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of avector. Large numbers of suitable vectors are known to those of skill inthe art and commercially available.

As used herein, “promoter” refers to a sequence of DNA, usually upstream(5′) of the coding region of a structural gene, which controls theexpression of the coding region by providing recognition and bindingsites for RNA polymerase and other factors which may be required forinitiation of transcription. The selection of the promoter will dependupon the nucleic acid sequence of interest. A suitable “promoter” isgenerally one which is capable of supporting the initiation oftranscription in a bacterium of the invention, causing the production ofan mRNA molecule.

As used herein, “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequence. A promoter sequence is “operably-linked” to a gene when it isin sufficient proximity to the transcription start site of a gene toregulate transcription of the gene.

“Percentage of sequence identity,” “% sequence identity” and “percentidentity” are used herein to refer to comparisons between an amino acidsequence and a reference amino acid sequence.

The “% sequence identify”, as used herein, is calculated from the twoamino acid sequences as follows: The sequences are aligned using Version9 of the Genetic Computing Group's GAP (global alignment program), usingthe default BLOSUM62 matrix (see below) with a gap open penalty of −12(for the first null of a gap) and a gap extension penalty of −4 (foreach additional null in the gap). After alignment, percentage identityis calculated by expressing the number of matches as a percentage of thenumber of amino adds in the reference amino acid sequence.

The following BLOSUM62 matrix is used:

Ala 4 Arg −1 5 Asn −2 0 6 Asp −2 −2 1 6 Cys 0 −3 −3 −3 9 Gln −1 1 0 0 −35 Glu −1 0 0 2 −4 2 5 Gly 0 −2 0 −1 −3 −2 −2 6 His −2 0 1 −1 −3 0 0 −2 8Ile −1 −3 −3 −3 −1 −3 −3 −4 −3 4 Leu −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 Lys−1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 Met −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5Phe −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 Pro −1 −2 −2 −1 −3 −1 −1 −2 −2−3 −3 −1 −2 −4 7 Ser 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 Thr 0 −1 0−1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 Trp −3 −3 −4 −4 −2 −2 −3 −2 −2−3 −2 −3 −1 1 −4 −3 −2 11 Tyr −2 −2 −2 −3 −2 −1 −2 −3 2 − −1 −2 −1 3 −3−2 −2 2 7 Val 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0 −3 −1 4 AlaArg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp TyrVal

“Reference sequence” or “reference amino acid sequence” refers to adefined sequence to which another sequence is compared. In the contextof the present invention a reference amino add sequence may, forexample, be an amino acid sequence set forth in SEQ ID NO: 5 or 6.

As used herein, “L-serine derivative” refers to a compound, such as anamino acid, resulting from reaction of L-serine at the amino group orthe carboxy group, or from the replacement of any hydrogen of L-serineby a heteroatom. Non-limiting examples of a “L-serine derivative”include L-cysteine, L-methionine, L-glycine, O-acetylserine,L-tryptophan, thiamine, ethanolamine and ethylene glycol. Furtherexamples of a “L-serine derivative” are described by Chemical Entitiesof Biological Interest (ChEBI) [https://www.ebi.ac.uk/chebi/init.do],for example, under ChEBI ID CHEBI:84135.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and sub ranges within a numerical limit orrange are specifically included as if explicitly written out.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples, which areprovided herein for purposes of illustration only, and are not intendedto be limiting unless otherwise specified.

EXAMPLES

For the first time, the present inventors show that a bacterium, such asE. coli, lacking all four serine degradation genes (sdaA, sdaB, tdcG andglyA) can be constructed (Example 1). Said strain shows higher serineproduction yield than single and triple deaminase knock outs, whenserine pathway is upregulated (Example 2). However, the resulting strainhad a low tolerance towards serine, which has also been reported in anE. coli triple deletion strain lacking sdaA, sdaB and tdcG (Zhang andNewman, 2008). The inventors furthermore demonstrate that producttoxicity can be reduced by over-expression of novel exporters (Example3), by evolving strains by random mutagenesis (Example 4), and byadaptive evolution (Example 5). The strain was furthermore reverseengineered in order to identify the causative mutations (Example 6).During fed batch fermentation, the tolerant strain shows improved serineproduction (12.6 g/L with a mass yield of 36.7% from glucose) whencompared to the parental quadruple deletion strain (Example 7). This isthe highest serine mass yield reported so far from glucose in anyproduction organism.

The inventors additionally demonstrate that inhibition of the pentosephosphate pathway by deletion of zwf in the presence of other causativemutations leads to a further increase in serine production yield(Example 8).

Example 1—Deletion of Key Degradation Pathways

Serine has two key degradation pathways in E. coli: Serine to pyruvate,which is encoded by three deaminases namely sdaA, sdaB and tdcG, andconversion of serine to glycine, which is encoded by glyA. The deletionof glyA renders E. coli auxotrophic for glycine. Deletion of sdaA, sdaBand tdcG was done sequentially by using the lambda red mediated genereplacement method (Datsenko and Wanner, 2000). The protocol applied fordeleting these genes is similar to the protocol described by Sawitzke etal. (2013). Primers used for amplification of the kanamycin cassette areshown in Table S1. The PCR reaction contained 250 nM each of KF and KRprimer of the given gene, 250 μM of dNTP mix, 4 Units of Phusionpolymerase (Thermosdentific, Waltham, Mass., USA), 40 μl of HF bufferand 10 ng of pKD4 plasmid. The following two-step PCR protocol was usedfor the PCR amplification: An initial denaturation step at 98° C. for 40seconds, followed by 5 cycles of denaturation at 98° C. for 10 seconds,annealing at 55° C. for 30 seconds, extension at 72° C. for 90 secondsthe cycle, followed by 20 cycles, where the annealing temperature wasincreased from 55° C. to 65° C. The PCR products were column purified(Macherey Nagel, Durn, Germany) and concentration was measured using aNanodrop instrument (Thermoscientific, Waltham, Mass., USA) andsubjected to overnight DpnI digestion. E. coli MG1655 was used as parentstrain to make sequential knock outs. The parent strain was transformedwith pKD46 were grown in 2YT-amp media at 30° C. and 250 rpm. Theexpression of exo, beta and gamma proteins were induced by addition of20 mM arabinose at mid log phase (O.D. 0.4 to 0.5) and the cells wereharvested after additional 1 h incubation. Culture was then transferredto 50 ml ice cold falcon tubes and centrifuged at 6500 rpm for 5 min at4° C. Supernatant was discarded and cells were washed twice with icecold 10% glycerol. These electro-competent cells were transformed with200 ng of kanamycin cassette, and transformants were plated on LB-kanplates. The kanamycin cassette was removed using the plasmid pcp20encoding flippase gene. Primers for checking the loop out are shown inTable 52. Serine hydroxymethyltransferase encoded by glyA was deletedusing the P1 phage system protocol (Thomason et al., 2007). A strainfrom the Keio collection (Baba et al., 2006) harboring glyA::kan wasgrown in 5 ml of LB-kan supplemented with 0.2% Glucose, 25 mM CaCl2 and1 mM Glycine media. At early log phase (O.D. 0.1), culture wastransduced with P1 phage lysate. The culture was incubated for threehours at 37° C. and 250 rpm for cell lysis. Lysate was filter sterilizedby using 0.45 uM filter. 1 ml of overnight culture of the E. coli strainhaving sdaA, sdaB and tdcG deleted, was resuspended in 200 μL of P1 saltsolution and was incubated with 100 μL of the above lysate for 1 h.Cells were then grown overnight in 2 mL LB media supplemented with 2 mMglycine and 200 mM sodium citrate. Cells were plated on LB-kan platesupplemented with 2 mM glycine and 10 mM sodium citrate. The clones wererestreaked onto a new plate in order to remove any phage contamination,and isolated colonies were checked for cassette insertion. The loop outwas done using pcp20 plasmid. The resulting quadruple deletion strain(FIG. 1) is referred to as Q1. This example demonstrates that it ispossible to delete sdaA, sdaB, tdcG and glyA in E. coli, something thathas not previously been achieved, and is thus unexpected.

TABLE S1 Primers used for amplification of kanamycin cassette Primername Sequence sdaA_KF GCGCTGTTATTAGTTCGTTACTGGAAGTCCAGTCACCTTGTCAGGAGTATTATCGTGGTGTAGGCTGGAGCTGCTTCG sdaA_KRCGCCCATCCGTTGCAGATGGGCGAGTAAGAAGTATTAGTCACACTGGACCATATGAATATCCTCCTTAGTTCC sdaB_KFCGCTTTCGGGCGGCGCTTCCTCCGTTTTAACGCGATGTATT GTCCTATGGTGTAGGCTGGAGCTCTTCGsdaB_KR GGCCTCGCAAAACGAGGCCTTTGGAGAGCGATTAATCGCAGGCAACCATATGAATATCCTCCTTAGTTCC tdcG_KFCGTTCCGCTCCACTTCACTGAACGGCAATCCGAGGGTGTGG ATATGGTGTAGGCTGGAGCTGCTTCGtdcG_KR GTGCACCCAAGGATGAAAGCTGACAGCAATGTCAGCCGCAGACCACCATATGAATATCCTCCTTAGTTCC glyA_KFGTTAGCTGAGTCAGGAGATGCGGATGTTAAAGCGTGAAATG AACATTGCCGTGTAGGCTGGAGCTGCTTCGglyA_KR CAACGAGCACATTGACAGCAAATCACCGTTTCGCTTATGCGTAAACCGGCATATGAATATCCTCCTTAGTTCC

TABLE S2 Primers used for checking the deletion of given genes Primername Sequence sdaA_cF GCGCTGTTATTAGTTCGTTACTGGAAGTCC sdaA_cRCGCCCATCCGTTGCAGATGGGC sdaB_cF CGCTTTCGGGCGGCGCTTCCTC sdaB_cRGGCCTCGCAAAACGAGGCCTTTGG tdcG_cF CGTTCCGCTCCACTTCACTGAACGG tdcG_cRGTGCACCCAAGGATGAAAGCTGACAGC glyA_cF GTTAGCTGAGTCAGGAGATGCGGATGTT glyA_cRCAACGAGCACATTGACAGCAAATCACCG

Example 2—Overexpression of Serine Biosynthesis Pathway for Productionof Serine

Serine is produced in E. coli from three enzymes encoded by serA, serBand serC. All genes were isolated from E. coli MG1655 using primers withrespective gene names (Table 53). The 100 μl PCR mixture contain 250 nMeach of forward and reverse primer, 250 μM of dNTP, 2 U of Phusionpolymerase, 1×HF buffer, 1 μl of overnight culture. The followingtwo-step PCR protocol was used for the PCR amplification: An initialdenaturation step at 98° C. for 40, followed by 5 cycles of denaturationat 98° C. for 10 seconds, annealing at 55° C. for 30 seconds, extensionat 72° C. for 90 seconds the cycle, followed by 20 cycles, where theannealing temperature was increased from 55° C. to 65° C. After columnpurification, the gene products and plasmids were digested using Fastdigest enzymes (Thermoscientific, Waltham, Mass., USA). About 500 ng ofPCR product or 1 μg of plasmids were subjected to digestion by 1 μl eachof the restriction enzymes in 1× fast digest buffer. The reaction wasincubated for 3h and then column purified again. serA was subjected todouble digestion with NcoI and NotI while serC was digested with NdeIand PacI. pCDF-Duet was first digested with NcoI and NotI for cloning ofserA leading to plasmid pCDF-Duet-serA and this plasmid was later usedfor cloning of serC thus making pCDF-Duet-serA-serC. The gene encodingserB was cloned in pACYC-Duet vector at NcoI and PacI site leading topACYC-serB. Typical ligation reaction include 1×T4 ligase buffer 50 ngof plasmid DNA and 100 ng of insert and 0.3 μl/10 μl of T4DNA ligase(Thermoscientific, Waltham, Mass., USA).

Feedback inhibition of serA was removed by mutating three residues H344,N346 and N364 to alanine (Al-Rabiee et al., 1996) by site directedmutagenesis (Table S4). The master mix was used as mentioned above withthe only modification that the master mix was divided in two equalaliquots, after which forward and reverse primers were added to eachaliquot. A total of 100 ng of pCDF-Duet-serA-serC plasmid was used as atemplate. The two step PCR program: initial denaturation at 98° C. for40 sec, denaturation at 98° C. for 10 sec, annealing 60° C. for 30 sec,extension 72° C. for 4 min and 30 sec the cycle was repeated 5 times andthen the two aliquots were mixed and redistributed for additional 15cycles. To enable swapping of vector backbones, the NcoI site insideserC was removed by the same approach using the primers listed in TableS4.

FIG. 2 shows the vector maps of the plasmid constructs.

TABLE S3 Primers used for amplification and cloning of serineproduction pathway Primer name Sequence serA-NcoIGGCCCATGGCAAAGGTATCGCTGGAG serA NotI_R ATTGCGGCCGCTTAGTACAGCAGACGGGCGCGAserB_NcoI_F GGCCCATGGCTAACATTACCTGGTGCG serB_R_HindIIIGCCAAGCTTTTATTACTTCTGATTCAGGCTGCC serB_PacI_RGCCTTAATTAATTATTACTTCTGATTCAGGCTGCC serC_F_NdeIGGCCATATGATGGCTCAAATCTTCAATTTTAGTTCTGG serC_R_PacIGCCTTAATTAATCATTAACCGTGACGGCGTTCGAAC ydeD_F_NdeICCGCATATGTCGCGAAAAGATGGGGTG ydeD_cHis_R-PacIGCCTTAATTAATGATGATGATGATGATGACTTC CCACCT TTACCGCTT TACGCC ydeD_Nco_F1CCGCCATGGCGCGAAAAGATGGGGTG

TABLE S4 Primers used for site directed mutagenesis ofserine production pathway Primer name Sequence serA N364A_FCGAGCAGGGCGTCGCTATCGCCGCGCAATA serA N364A_RTATTGCGCGGCGATAGCGACGCCCTGCTCG serA H344AN346A_FCTGATCACATCGCTGAAGCTCGTCCGGGCG TGC serA H344AN346A_RGCACGCCCGGACGAGCTTCAGCGATGTGCA TCAG serC_Ncolc_FCAAGGTATTATTCTGTCATGGCGGTGGTCG CG serC_Ncolc_RCGCGACCACCGCCATGACAGAATAATACCT TG

DE3 Integration and Serine Production During Batch Fermentation

The serine production was checked in M9 minimal media. Glucose M9minimal media consisted of 2 g/L glucose, 0.1 mM CaCl₂, 2.0 mM MgSO₄, 1×trace element solution, and 1×M9 salts. The 4,000× trace element stocksolution consisted of 27 g/L FeCl₃*6H₂O, 2 g/L ZnCl₂*4H₂O, 2 g/LCoCl₂*6H₂O, 2 g/L NaMoO₄*2H₂O, 1 g/L CaCl₂*H₂O, 1.3 g/L CuCl₂*6H₂O, 0.5g/L H₃BO₃, and Concentrated HCl dissolved in ddH₂O and sterile filtered.The 10× M9 salts stock solution consisted of 68 g/L Na₂HPO₄ anhydrous,30 g/L KH₂PO₄, 5 g/L NaCl, and 10 g/L NH₄Cl dissolved in ddH₂O andautoclaved.

To use pET vectors as expression system, a DE3 cassette containing T7polymerase was integrated into the genome of each deletion mutant usinga Lambda DE3 lysogenization kit (Millipore, Damstadt Germany).Subsequently, strains (MG1655 (DE3)) carrying single (ΔsdaA), triple(ΔsdaA, ΔtdcG, ΔsdaB), and quadruple deletions (ΔsdaA, ΔtdcG, ΔsdaB andΔglyA) were transformed with pCDF-Duet1-serAmut-serC and pACYC-serB. Theresulting glycerol stocks were grown overnight in 2YT medium containing0.1% glucose and supplemented with required antibiotics (Spectinomycinand chloramphenicol). Overnight cultures were inoculated in triplicatesinto flasks containing M9 medium supplemented with 0.2% glucose, 1 mMthreonine, required antibiotics (For the quadruple deletion strain, 2 mMglycine was also supplemented). Flasks were incubated at 37° C. at 250rpm. Serine production was induced by addition of 40 μM IPTG after thecultures reached an optical density of 0.55 to 0.65. O.D. measurementsand sampling were done at regular time intervals for following 60 hr.Samples were filtered and subjected to HPLC for glucose and byproductanalysis using a method described previously (Kildegaard et al., 2014)with the only exception that the column temperature was kept at 30° C.Serine concentrations were determined using LCMS. The LC-MS/MS systemconsisted of a CTC autosampler module, a high pressure mixing pump and acolumn module (Advance, Bruker, Fremont, Calif., USA). The injectionvolume was 1 μl. The chromatography was performed on a ZIC-cHILICcolumn, 150 mm×2.1 mm, 3 μm pore size, (SeQuant, Merck Millipore). Infront of the separation column was a 0.5 p depth filter and guardcolumn, the filter (KrudKatcher Classic, phenomenex) and guard columnZIC-cHILIC, 20×2.1 mm(SeQuant, Merck). Eluent A: 20 mM ammonium acetatepH adjusted to 3.5 with formic acid in milliQ water. Eluent B:Acetonitrile. The total flow rate of eluent A and B was 0.4 ml/min. Theisocratic elution 35%, and the total run time was 5 minutes. Retentiontime was 2.8 minutes for serine. The MS-MS detection was performed on aEVOQ triple quadrupole instrument (Bruker, California, USA) equippedwith an atmospheric pressure ionization (API) interface. The massspectrometer was operated with electrospray in the positive ion mode(ESI+). The spray voltage was set to 4500 V. The con gas flow was 20l/h, and the cone temperature was set at 350° C. The heated probe gasflow was set at 50 l/h with a temperature of 350° C. Nebulizer flow wasset at 50 l/h, and the exhaust gas was turned on. Argon was used ascollision gas at a pressure of 1.5 mTorr. Detection was performed inmultiple reacting monitoring (MRM) mode. The quantitative transition was106→60, and the qualitative transition was 106→70. The collision energywas optimized to 7 eV for both transitions. Calibration standards ofserine were prepared in media used for serine production and diluted50:50 with 0.2% Formic acid in Acetonitrile. The concentration of thecalibration standards was in the range from 0.001 to 0.5 g/L

The experiment demonstrates that the deletion of all four genes involvedin serine degradation in E. coli, referred to as Q1(DE3), results in thehighest specific productivity and the highest yield from glucose asshown on FIG. 3.

Example 3: Reducing Product Toxicity by Overexpression of Transporter

Deletion of the main serine degradation pathways in E. coli results insignificant decreased tolerance towards serine. In order to increase thetolerance, a transporter with a potential specificity for serine (ydeD)was overexpressed and tested during growth in high concentrations ofserine. ydeD was cloned in pCDF-1b at NcoI and PacI site byamplification with primers mentioned in Table S3 and the protocol givenin Example 1.

Q1(DE3) was transformed with the pCDF-1b empty vector and thepCDF-ydeD-c-His plasmid (FIG. 4). Transformants were selected onLB-spectinomycin plates. Overnight cultures of these transformants wereinoculated in M9 media supplemented with 2 mM glycine, 2 g/L glucose andspectinomycin in 1:50 ratio. Cultures were incubated at 37° C. and 250rpm at an optical density of 0.4 to 0.5, after which 800 μl was added toa 48-well biolector plate (M2P labs, Baeswieler, Germany) containing 800μl M9 media with varying serine concentration (12.5, 25 and 50 g/L) and2 mM glycine. The expression of ydeD was induced with 100 μM IPTG. Thegrowth was then monitored in the Biolector instrument (M2P labs,Baeswieler, Germany) at 37° C. and 70% humidity with continuous shaking.Gain was set to 20% and scattering intensity was measured every 5 minfor next 40 h.

The experiment demonstrates that the growth of E. coli lacking the mainserine degradation pathways are severely growth inhibited in thepresence of even low concentrations of serine. Upon overexpression ofydeD, the tolerance towards serine is increased substantially (FIG. 4),suggesting that YdeD may potentially transport serine out of the cell.

Example 4—Random Mutagenesis for Serine Tolerant Strain

Inactivation of the main serine degradation pathways in E. coli resultsin significant decreased tolerance towards serine. In order to increasethe tolerance, the Q1 strain (obtained in Example 1) was grown in M9media supplemented with 2 mM glycine and 3 g/L serine overnight. 1 ml ofculture was spread in 6 well petri plate and exposed to UV irradiation(CBS Scientific, San Diego, Calif., USA) for 30 min. The culture wasthen added to 5 ml M9 media supplemented with 2 mM glycine, 2 g/Lglucose and 50 g/L serine for enrichment of the tolerant mutants. Theculture was incubated at 37° C. and 250 rpm for 3 days and then platedon M9 plate supplemented with 50 g/L serine for the selection of thetolerant clones. Colonies were isolated and the growth rates andtolerance was estimated using the following method:

Cultures were grown overnight in 2×YT media. They were then inoculatedin 96 well flat bottom micro titer plates with 150 μL of M9 mediacontaining 0.2% glucose and various concentration of Serine (intriplicates). For strains containing the glyA deletion, 2 mM glycine wasalso supplemented to the media. A total of 1.5 μL of cells (1:100) wasused as inoculum. If required minor changes is volume were done toensure equal amount of cells. Plates were sealed with Microamp clearadhesive film (Applied Biosystems, Warrington, UK) and were incubated inMicrotiter plate reader (Biotek, Winooski Vt., USA). The reader was setat 37° C. with continuous shaking, and the O.D was monitored every 5 to10 minutes at 630 nm for 32 hours.

The experiment demonstrates that random mutagenesis can be used toincrease the tolerance towards serine significantly (FIG. 5). Theresulting strain is referred to as Q3 below.

Example 5—Adaptive Evolution for L-Serine Tolerance

Apart from random mutagenesis, serine tolerance of a strain having themain serine degradation pathways inactivated was also increased byAdaptive evolution. Seven independent populations of the Q1 strain wereadaptively evolved in M9 minimal media supplemented with 2 mM glycineand increasing concentrations of the amino acid L-Serine at 37° C. along60 days.

An Adaptive Laboratory Evolution (ALE) experiment was achieved by usingan automated system, which enables the propagation of evolvingpopulations over the course of many days while monitoring their growthrates. Prior to the start of the experiment, the system filled therequired tubes with 25 ml of culture media and kept them at 37° C. in aheat block. Controlled aeration was obtained using magnetic tumblestirrers placed inside the tubes and spinning at 1,800 rpm. At the startof the experiment, a single colony was grown overnight in one of thetubes placed inside the system, and 100 μL aliquots were used by therobotics platform to inoculate seven independent flasks. As the bacteriagrew, the automated system performed multiple OD measurements at 600 nmfor each flask. Growth rates were automatically calculated by taking theslope of a least-square linear regression line fit to the logarithm ofthe OD measurements (FIG. 6). Once reaching a target OD of 0.4, 100 μlof culture was used to inoculate a new flask. This way, cultures wereserially passed (˜2-3 times per day) to flasks with fresh media afterreaching a targeted cell density such that stationary phase was neverreached. The populations were initially incubated with 3 g/L serineafter desired growth rate was reached culture was supplemented with 6g/L of L-Serine. When populations achieved a stable phenotype (i.e.growth rate), the L-Serine concentration was increased to 12 g/L. Thisprocess was repeated iteratively using 24, 50, 75, and 100 g/L ofL-serine. The final population was plated on the LB-agar and 2 clones ofeach of the populations selected. The evolved strains had significantlyincreased tolerance when tested for growth in the presence of highconcentrations of serine by using the MTP based assay described inExample 5, above. The results are shown in FIG. 6.

Genomic DNA Extraction, Library Sequencing and Analysis

Two clones of each evolved cultures as well as the strain evolved byrandom UV-mutagenesis (Example 5) were genome sequenced. Genomic DNA wasextracted from 1.5 ml of overnight cultures of E. coli strains usingQIAamp DNA Mini Kit (QIAGEN, Germany). The genomic libraries weregenerated using the TruSeq®Nano DNA LT Sample Preparation Kit (IlluminaInc., San Diego Calif., USA). Briefly, 100 ng of genomic DNA diluted in52.5 ul TE buffer was fragmented in Covaris Crimp Cap microtubes on aCovaris E220 ultrasonicator (Covaris, Brighton, UK) with 5% duty factor,175 W peak incident power, 200 cycles/burst, and 50-s duration underfrequency sweeping mode at 5.5 to 6° C. (Illumina recommendations for a350-bp average fragment size). The ends of fragmented DNA were repairedby T4 DNA polymerase, Klenow DNA polymerase, and T4 polynucleotidekinase. The Klenow exo minus enzyme was then used to add an ‘A’ base tothe 3′ end of the DNA fragments. After the ligation of the adapters tothe ends of the DNA fragments, DNA fragments ranging from 300-400 bpwere recovered by beads purification. Finally, the adapter-modified DNAfragments were enriched by 3-cycle-PCR. Final concentration of eachlibrary was measured by Qubit® 2.0 Florometer and Qubit DNA Broad rangeassay (Life Technologies, Paisley, UK). Average dsDNA library size wasdetermined using the Agilent DNA 7500 kit on an Agilent 2100Bioanalyzer. Libraries were normalized and pooled in 10 mM Tris-Cl, pH8.0, plus 0.05% Tween 20 to the final concentration of 10 nM.Denaturated in 0.2N NaOH, 10 pM pool of 20 libraries in 600 μl ice-coldHT1 buffer was loaded onto the flow cell provided in the MiSeq Reagentkit v2 300 cycles and sequenced on a MiSeq (Illumina Inc., San Diego,Calif., USA) platform with a paired-end protocol and read lengths of 151nt.

The breseq pipeline (Deatherage and Barrick, 2014) version 0.23 withbowtie2 (Langmead and Salzberg) was used to map sequencing reads andidentify sequence variants relative to the E. coli K12 MG1655 referencegenome (NCBI accession number NC_000913.2). Gene deletions present inthe strains were verified manually based on missing coverage regions inthe genome. Common variants found in MG1655 stock cultures (Freddolinoet al., 2012) were excluded from further analysis. All sequencingsamples had an average mapped coverage of at least 25×. The experimentidentifies mutations that cause increased tolerance towards highconcentrations of serine as shown in Table S5.

TABLE S5Mutations found in the strains evolved for increased serine tolerance.The reference number for the genome sequence is NC_000913. ALE5-  ALE5-ALE6- ALE6- Gene Name Change Annotation Q1 Q3 4 8 1 2 thrA A -> G Y356C0 0 0 0 0 0 thrA A -> C S357R 0 0 1 1 0 0 thrA T -> A S359R 0 0 0 0 1 1trxA/rho IS1 -1 9 bp Insertion¹ 0 0 0 0 0 0 rho G -> T R87L 0 0 0 0 1 1gcvA/ygdl 1 bp Insertion² 0 0 0 0 1 1 gcvA/ygdl 1S4 1 13 bp Insertion³ 00 1 1 0 0 dapA/gcvR 1 bp Insertion⁴ 0 0 1 0 1 1 lrp A -> G D143G 0 0 0 00 0 trxB/lrp C -> T Intergenic⁹ 0 0 0 0 1 1 (−271/−274) frc IS1 -1 9 bpInsertion⁵ 0 0 0 0 0 0 eno C -> G V164L 0 0 0 0 0 0 argP C -> A Q132K 00 0 0 0 0 tufA C -> A G19V 0 0 0 0 0 0 cycA A -> G 1220V 0 0 0 0 0 0rhtA/ompX/ybiP/mntS 2854 bp Deletion⁶ 0 1 0 0 0 0 rpe A -> G I202T 0 0 00 1 1 ytfB/fklB T -> C Intergenic¹⁰ 0 0 0 0 1 1 (-154/-64) yojL G --> CD334H 0 0 0 0 1 0 aroP IS5 1 4 bp Insertion⁷ 0 0 0 1 1 0 hyaF T -> GV120G 0 0 1 1 0 0 mdtJ/tqsA IS1 -1 9 bp Insertion⁸ 0 0 0 0 0 0 pykF G ->T E250* 0 0 1 1 0 0 malT C -> T Q420* 0 0 1 1 0 0 rpoB C -> T P520L 0 01 1 0 0 fumB T -> G 1218P 0 0 0 0 0 0 gshA G --> A A178V 0 0 0 1 0 0lamB C --> T Q112* 0 0 0 1 0 0 ALE8-  ALE8- ALE9- ALE9- Gene Name ChangeAnnotation Q1 3 8 3 8 thrA A -> G Y356C 0 1 1 1 1 thrA A -> C S357R 0 00 0 0 thrA T -> A S359R 0 0 0 0 0 trxA/rho IS1 -1 9 bp Insertion¹ 0 1 11 1 rho G -> T R87L 0 0 0 0 0 gcvA/ygdl 1 bp Insertion² 0 0 0 0 0gcvA/ygdI IS4 1 13 bp Insertion³ 0 0 0 0 0 dapA/gcvR 1 bp Insertion⁴ 0 00 0 0 lrp A -> G D143G 0 1 1 1 1 trxB/Irp C -> T Intergenic⁹ 0 0 0 0 0(−271/−274) frc IS1 -1 9 bp Insertion⁵ 0 1 1 1 1 eno C -> G V164L 0 1 11 1 argP C -> A Q132K 0 1 1 1 1 tufA C -> A G19V 0 1 1 1 1 cycA A -> G1220V 0 1 1 1 1 rhtA/ompX/ybiP/mntS 2854 bp Deletion⁶ 0 0 0 0 0 rpe A ->G I202T 0 0 0 0 0 ytfB/fklB T -> C Intergenic¹⁰ 0 0 0 0 0 (−154/−64)yojl G --> C D334H 0 0 0 0 0 aroP 1S5 1 4 bp Insertion⁷ 0 0 0 0 0 hyaFT -> G V120G 0 0 0 0 0 mdtJ/tqsA IS1 -1 9 bp Insertion⁸ 0 0 0 0 1 pykFG -> T E250* 0 0 0 0 0 malT C -> T Q420* 0 0 0 0 0 rpoB C -> T P520L 0 00 0 0 fumB T -> G T218P 0 0 0 1 0 gshA G- -> A A178V 0 0 0 0 0 lamBC- -> T Q112* 0 0 0 0 0 *designates a stop codon

Insertion¹: Insertion of a 768 bp long insertion sequence element IS1 Inthe lagging strand at the location 3966174, which is an intergenicregion between genes trxA and rho. Duplication of 9 bp upstream anddownstream of insertion sequence is observed.

Insertion²: Insertion of 1 bp at location 2942629, which is anintergenic region between genes genes gcvA and ygdI.

Insertion³: Insertion of 1342 bp long insertion sequence element 154 atthe location 2942878, which is an intergenic region between genes gcvAand ygdI. Duplication of around 13 bp upstream and downstream ofinsertion sequence is observed.

Insertion⁴: Insertion of 1 bp at location 2599854, which is anintergenic region between genes genes dopA and gcvR.

Insertion⁵: Insertion of 768 bp long insertion sequence element IS1 Inthe lagging strand at location 2492323, which leads to a truncation ofgene frc. Duplication of 9 bp upstream and downstream of insertionsequence is observed.

Deletion⁶: Deletion of 2854 bp from location 850092, resulting indeletion of the first 5 bp of rhtA, complete deletion of genes ompX andybiP, deletion of 239 bp of sRNA rybA and 77 bp deletion of mnt5 gene.

Insertion⁷: Insertion of 1195 bp long insertion sequence element IS5 atlocation 121518 which leads to deletion of the majority of aroP.Duplication of 4 bp upstream and downstream of insertion sequence isobserved.

Insertion⁸: Insertion of 768 bp long insertion sequence element 151 inthe lagging strand at location 1673670, which is an intergenic regionbetween genes mdtU and tqsA. Duplication of 9 bp upstream and downstreamof insertion sequence is observed.

Intergenic⁹: C→T mutation at location 923321 which is an intergenicregion between trxB and Irp. The mutation is 271 bp upstream of of trxBand 274 bp upstream of Irp.

Intergenic¹⁰: T→C mutation at the location 4428871 which is anintergenic region between yftB and fklB. The mutation is 154 bp upstreamof yftB and 64 bp upstream of fklB.

Example 6—Reverse Engineering of ALE Mutations by Cam-sacB System andMultiplex Genome Engineering

In order to identify mutations that cause increased tolerance towardsserine, the mutations identified in Example 5 were introduced into theQ1(DE3) strain using two different methods as described below.

A. Introduction of thrA Mutations

Mutations in thrA were introduced into the genome of the Q1(DE3) strainusing a cam-sacB based selection system. Cam-sacB was inserted usingpKD46 plasmid harboring exo, beta and gamma genes for recombination.Positive selection for cassette insertion was done by selecting clonesfor chloramphenicol resistance. The loss of cassette was selected byreplica plating clones on LB-chloramphenicol and LB-sucrose platecontaining 15% sucrose (no NaCl). The Cam-sacB cassette was amplifiedusing thrA_camsacB_F and R (Table S6) primers respectively. Apart fromtemplate and extension time, the reaction mixture and PCR program wasthe same as described in example 1. The extension time was 2 min and 30sec, while template was 1 μl of overnight culture of the E. coli culturecontaining cam-sacB cassette. Competent Q1(DE3) cells were thentransformed with 200 ng of thrA-cam-sacB cassette, and were plated onLB-chloramphenicol-ampicillin plates after two hour and incubatedovernight at 30° C. A single colony was picked and made electrocompetentafter 1 h of induction (Example 1) and transformed with thrA alleles.After 2 hours of recovery, cells were plated on LB-sucrose plates andincubated at 37° C. to cure pKD46 plasmid. 24 clones of each experimentwere replica plated on LB-chloramphenicol and LB plates. Clones that didnot grow on chloramphenicol plates were checked for the loss of thecassette by colony PCR and were subsequently sequenced by Sangersequencing.

All three strains containing thrA mutations (Y356C, S357R or S359R) weregrown in M9 media supplemented with 2 mM glycine, 2 g/L glucose and 6.25g/L L-serine. In this experiment, the background was not subtracted fromthe measurements, resulting in apparent increase in the OD for the Q1strain. However, under these conditions, the Q1 strain did not showsignificant growth. On the other hand, each of the thrA mutationsresulted in a similar and very significant increase in tolerance towardsL-serine (FIG. 7), allowing them to grow at a L-serine concentration ofat least 6.25 g/L opposed to the parent Q1 strain. Since all thrAmutants had same growth profile, S357R mutant was chosen as the parentstrain (referred to as strain Q1(DE3)-thrAS357R) for the multiplexgenome engineering described below.

TABLE S6 Primers used for genome integration of thrAmutations in the genome. Primer name Sequence thrA_gFATGCGAGTGTTGAAGTTCGGCG thrA_gR TCAGACTCCTAACTTCCATGAGAGGG thrA_camsacB_FATGCGAGTGTTGAAGTTCGGCGGTACATCAGTGG CAAATGCAGAACGTTTAAAATGAGACGTTGATCGGCACG thrA_camsacB_R TCAGACTCCTAACTTCCATGAGAGGGTACGTAGCAGATCAGCAAAGACACCAAAGGGAAAACTGTCCA TATGCAC

B. Multiplex Genome Engineering, Screening and Amplicon Sequencing ofSelected ALE Mutations

In order to identify mutations that cause increased tolerance towardsserine, selected mutations identified in Example 5 were introduced intothe Q1(DE3)-thrAS357R strain using multiplex genome engineering (Wang etal., 2011). The protocol applied for multiplex genome engineering wassimilar to the method published by Wang and Church, 2011. StrainQ1(DE3)-thrAS357R was transformed with plasmid pMA1 (containing onlybeta protein under control by the arabinose inducible promoter). Cloneswere plated on LB-ampicillin plates and incubated at 37° C. Colonieswere cultured overnight in TY-amp-gly media at 37° C. and re-inoculatedin 25 ml fresh TY-gly media the following day. After reaching an O.D. of0.4 at 37° C., arabinose was added to a final concentration of 0.2%.Cells were re-incubated at 37° C. and 250 rpm for additional 15 min andwere then made electro-competent by washing twice with 10% glycerol (icecold). Final volume of electro competent cells was adjusted to 200 μlusing 10% glycerol. The primers targeting the loci (Irp D143G, enoV164L, argP Q132K, cycA 1220V, pykF E250′, rpoB520L, gcvA*, yojL D334H,rho R87L) are given in Table 57. All primers were pooled and adjusted toa final concentration of 10 pmol/μl. To 50 μl of cells, 1 μl of this mixwas added and transformed by electroporation. The transformed cells weredirectly added to 25 ml TY-gly media and were incubated at 37° C., 250rpm for two hours to reach O.D. of 0.4, followed by arabinose inductionand transformation as above for second round of multiplex engineering.The process was repeated six times. After the 6th round of multiplexgenome engineering, the cells were incubated overnight. The resultinglibrary of mutants was subsequently grown in M9 minimal media with andwithout serine. This way, mutations resulting in increased growth ratesin minimal media or increased tolerance towards serine were enriched: Atotal of 1.5 μl of overnight culture was added in triplicates to wellsin micro titer plates containing 150 μl triplicates of M9 mediasupplemented with 2 mM glycine and 2 g/L glucose (minimal media) or M9media with 2 mM glycine, 2 g/L glucose and 25 g/L serine (minimal mediawith serine). Growth profiles were monitored at 37° C. with continuousfast shaking in a microtiter plate reader for 40 hr. Optical density wasmeasured at 630 nm every 5 min. Triplicates were pooled and used astemplate for amplicon generation. The PCR program and reactioncomposition was as described in Example 1. Illumina protocol wasfollowed for amplicon processing and next gen sequencing was asdescribed in Example 5. Sequencing results were analyzedcomputationally, and the enrichment of the various mutations wascalculated as the frequency of the mutations in either minimal media orminimal media with serine, divided by the frequency of the mutations inthe unselected library (FIG. 8). This experiment shows that themutations in Irp, rho, argP and eno results in increased tolerancetowards serine.

TABLE S7 Primers used for Multiplex genome engineering Primer nameSequence IrpD143GGCTTGACTTCTTCCATAACAACGTAAGTGCGCGTCCCGTTAACCCCAGGCAGACGC AGCAGGGTTTCenoV164L CCAACCGGCTGAATCATGAATTCTTGAATGTCGAGATTATTATCAGCGTGCTCACCACCGTTGATGATG argPQ132KCTCAACTTGCAGGTAGAAGATGAAACGAGGAAAGAGAGGCTCCGCCGCGGCGA AGTGGTCGGCcycAI220V CAGCTCAATCCCCACGAAAGCTAATACGGCAACTTGAAAACCGGCAAAGAAGCCACTTAAACCTTTC pykFE250*** GCATCATGGTTGCGCGTGGCGACCTCGGCGTTTAGTGACCCTAAATCTTCGCCCAGAAGATGATGA rpoBP520LGATACGACGTTTGTGCGTAATTTCTGAGAGGAGATTATTTTGGTCCATAAACTGA GACAGCTG gcvA***CTCGTAAGGCATTTAGCGGTGGTAATGGTTATC ATTAGGCTATTAAACTTTGATGTTAAATGyojLD334H GGTGAGATTAATCGGACCAACGGAGAAGGCATTGTGTTGGTATGCAAACGTCACGTTACGCAGCTCCAGC rhoR87LGAGATGGTATCACCAGTGCGGAGATTAAATCTTAGTATCTGAGAAGGGGAAACG TAGATGTCATCAG

TABLE S8 Primers used for amplicon sequencing Primer name Sequence Irp_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCGGTGATTTCGACTACCTGTTG Irp_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCGCGTCTTAATAACCAGACGAT enoV164_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCTGTACGAGCACATCGCTGAAC enoV164_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCCATGCGGATGGCTTCTTTCAC argP_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGACAGTCTGGCGACGTGGTTGCT argP_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTATCGACAAGACAACTCGGCAGCG cycA_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGAAGCGTCATTCGCGCATTTG cycA_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTAATCGCGCGTGGCAGTG pyfK_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGCCTCAACAACTTCGACGA pyfK_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAATCCAGCATCTGGGTCGC rpoB_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAACGCCAAGCCGATTTCCG rpoB_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTCTCGAACTTCGAAGCCTGC gcvA_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCTGGTAGAAGCTCAACGGAC gcvA_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGCTGCGGCATCAAAAACTCG yoji_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCCTTTCAAAGCAGAGTTTCCGC yoji_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTACCGTTGCCGCCAATCAG rho_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCAGGATGGATTTGGTTTCCTCC rho_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCAGCGCAAAATAGCGTTCACC

Example 7—Serine Production by Tolerant and Susceptible E. coli DuringFed Batch Fermentation

A new vector was constructed in order to upregulate expression ofexporter along with serine pathway in the pACYC-Duet vector by followingthe same above strategy as mentioned in example 2. serB was cloned usingNcoI and HindIII double digestion followed by ligation. The resultingvector (pACYC-Duet-serB) was then subjected to double digestion by NdeIand PacI for cloning of ydeD-c-His. Both strains Q1 (DE3) and Q3 (DE3)were made electrocompetent by growing cells in 30 ml 2×YT mediasupplemented with 0.2% glucose (37° C., 250 rpm) to mid log phase. Cellswere harvested by centrifugation at 6500 rpm for 5 min at 4° C., andwere washed twice by ice cold 10% glycerol. Cells were finallyresuspened in 500 μL of 10% glycerol and 50 μL of cells were transformedwith plasmids pCDF-Duet1-serAmut-serC and pACYC-serB-ydeD-c-His forserine production.

Serine production was demonstrated during fed batch fermentation in 1 Lfermenters (Sartorius, Gottingen, Germany). The media contained 2 g/LMGSO4.7H2O, 2 g/L KH2PO4, 5 g/L (NH4)2SO4, 7.5 g/L Glucose, 2 g/L Yeastextract, 0.6 g/L glycine, 0.12 g/L threonine, 4× trace element (asmentioned in Example 2), 50 mg/L spectinomycin and 25 mg/Lchloramphenicol. For the E. coli Q1(DE3) strain, the initial glucoseconcentration was 10 g/L in order to enhance the growth beforeinduction. The feed generally contained 140 g/L glucose, 24 g/L Ammoniumsulfate, 2 g/L glycine, 0.12 g/L threonine, 2.5 g/L each of MGSO4*7H2Oand KH2PO4, 1× trace elements 50 mg/L spectinomycin and 25 mg/Lchloramphenicol, while the feed for the Q1(DE3) strain contained 120 g/Lof glucose.

A log phase culture was used to inoculate 500 ml media by 1:50 inoculumratio. The cultures were allowed to grow over night in the fermenter at37° C., 1000 rpm baffle speed and 20% air saturation. Feed at the rateof 8 g/h was started after glucose concentration was below 250 mg/L inthe media. Production was induced at late log phase (O.D. 7.5 to 9.5) bythe addition of 40 μM IPTG and feed rate was reduced to 6 g/h. Sampleswere taken at regular intervals and were subjected to HPLC and LCMSanalysis as mentioned before.

The experiment demonstrates that serine can be produced at high titerand with a high yield in E. coli using fed batch fermentation (FIG. 9).The titers were 6.8 g/L and 12.5 g/L for the Q1(DE3) and the Q3(DE3)strain, respectively. The fermentations resulted in a surprisingly highmass yield of 36.7% from glucose in the Q3(DE3) strain when compared to24.3% in the Q1(DE3) strain (FIG. 9B). This shows that a higher titerand yield can be achieved using the production strain that is toleranttowards serine.

Example 8—Reverse Engineered Strains Showing Increased Serine Production

Strains reverse engineered in example 6 were checked for serinetolerance as mentioned in example 3. The best serine tolerant strain(F7) was checked for serine production and was also genome sequenced asdescribed in example 5. Plasmids for overexpression of the serineproduction pathway were introduced as described in Example 7. Shakeflask experiments were carried out as described in Example 2, with theonly difference being that the concentration of glucose was 2.5 g/L. TheO.D. measurements and sampling were done at regular intervals, and thesamples were analyzed using methods described in Example 2.Surprisingly, said strain resulted in a significantly increasedproduction and yield of serine from glucose as shown in Table 59. Thegenome sequence revealed a deletion of zwf, which encodes the firstenzyme in the pentose phosphate pathway. Additionally the strain carriedthe thrAS357R and rhoR87L mutations. Furthermore the strain also had atruncation of the branched chain amino acid exporter brnQ (105 bp wasdeleted starting at location 419986. The 439 amino add protein wastruncated after 308 amino acids).

TABLE S9 Effect of Δzwf on production of serine Serine concentration ing/L Yield from glucose (+/−standard diviation) (g serine/g glucose)ΔsdaA ΔsdaB 0.925 (+/−0.048) 0.37 ΔtdcG ΔglyA (Q1) ΔsdaA ΔsdaB ΔtdcG1.323 (+/−0.06)  0.53 ΔglyA Δzwf Δbrnq thrA S357R rho R87L

Example 9—Production by ALE 8

To use pET vectors as expression system, a DE3 cassette containing T7polymerase was integrated into the genome of the ALE 8-8 mutant (Example5) using a Lambda DE3 lysogenization kit (Millipore, Damstadt Germany)resulting in the strain ALE 8-8(DE3). Subsequently, the ALE 8-8(DE3) wasmade electrocompetent by growing cells in 30 ml 2×YT media supplementedwith 0.2% glucose (37° C., 250 rpm) to mid log phase. Cells wereharvested by centrifugation at 6500 rpm for 5 min at 4° C., and werewashed twice by ice cold 10% glycerol. Cells were finally resuspended in500 μL of 10% glycerol and 50 μL of cells were transformed with plasmidspCDF-Duet1-serAmut-serC and pACYC-serB for serine production.

Serine production was demonstrated during fed batch fermentation in 1 Lfermenters (Sartorius, Gottingen, Germany) as mentioned in example 7.The media contained 2 g/L MGSO4*7H2O, 2 g/L KH2PO4, 5 g/L (NH4)2SO4, 10g/L Glucose, 2 g/L Yeast extract, 0.6 g/L glycine, 4× trace elements (asmentioned in Example 2), 50 mg/L spectinomycin and 25 mg/Lchloramphenicol. The 375 g of feed contained 400 g/L glucose, 24 g/LAmmonium sulfate, 2 g/L glycine, 2.5 g/L of MGSO4*7H2O and 5 g/L ofKH2PO4, 1× trace elements 50 mg/L spectinomycin and 25 mg/Lchloramphenicol. A log phase culture was used to inoculate 500 ml mediaby 1:50 inoculum ratio. The cultures were allowed to grow overnight inthe fermenter at 37° C., 1000 rpm baffle speed and 20% air saturation.Production was induced at late log phase (O.D. 8.5 to 9.5) by theaddition of 80 μM IPTG and feed at the rate of 8 g/h was started.Samples were taken at regular intervals and were subjected to HPLC andLCMS analysis as mentioned before.

The experiment shows that both a high cell density and a high serinetiter (55.0 g/L) can be obtained in ALE 8-8 (DE3) strain (FIG. 10A) witha mass yield from glucose of 36.0% (FIG. 10B).

Example 10—Over Expression and Site Saturation Mutagenesis of thrA atSite Y356, S357 and S359

Using site saturation mutagenesis (SSM), the selected amino acidresidue/s can be mutated to all other 20 amino acids. This makes itpossible to investigate the effect of amino acid substitutions on theenzyme activity and in turn on phenotype. The thrA gene was amplifiedfrom MG-1655 wt strain using primers thrA_NcoI_F and thrA_HindIII(primer sequences in Table 510) and cloned in pACYC-Duet1 plasmid usingNcoI and HindIII enzyme. The protocol of cloning was same as for cloningserB in example 2. The vector constructed was pACYC-thrA. This plasmidwas used as a template for SSM. SSM for observed thrA mutations in ALEstrains (Y356, S357 and S359) was performed using primers given in Table510. The SSM protocol was similar to SDM protocol applied in Example 2for mutagenesis of feedback insensitive serA. PCR product was DpnIdigested and transformed in Q1 (DE3) strain, which carries the wild typethrA gene in its genome, and plated on LB-chloramphenicol platessupplemented with 0.1% glucose and 4 mM glycine. Chloramphenicol wassupplemented in all following media for plasmid stability.

Individual colonies were picked and inoculated into 96 well MTP platecontaining 2× TY media supplemented with 0.1% glucose and 2 mM Glycine.90 clones (1 microtiter plate (MTP)) from each SSM library was picked(in total 3 plates). The Q1(DE3) strain harboring pACYC-Duet emptyvector and pACYC-thrAwt was inoculated as a control strain in eachplate. The plate was incubated at 37° C. and 300 rpm overnight. Thispre-culture plate was used as inoculum from which 1 μL was added to anew 96 well MTP plate containing 100 μL of M9 media supplemented with0.2% glucose, 2 mM glycine. The culture was incubated for 4 h afterwhich 25 uL of 1 mM IPTG was added to induce the expression. The platewas subsequently incubated for 2 h and then M9 media containing 0.2%glucose, 2 mM glycine and 12.5 g/L L-serine was added thus reachingfinal serine concentration of 6.25 g/L The plate was then incubated withshaking in an MTP reader for growth analysis as described before.Selected clones showing tolerance from each plate were collected in 1plate and the tolerance studies were repeated for these strains. Theplasmids from the tolerant cloned were sequenced and the growth rateswere estimated from the log phase of culture. This way, a number ofamino acid substitutions that result in tolerance towards serine wereidentified (FIG. 11). FIG. 11 (A-C) shows the growth rate of mutantstrains having different amino acid substitutions observed at positions356, 357 and 359 of ThrA, respectively (denoted by the respectiveone-letter code). The growth rate of E. coli carrying the wild type thrAgene is denoted “wt”.

The experiment demonstrates that amino acid substitutions at position356, 357 and/or 359 in the native ThrA protein confer increasedtolerance of modified strains towards L-serine. The data furtherdemonstrates that overexpression of mutants of ThrA can increase thetolerance towards L-serine even in strains having a native thrA genepresent in their genome.

TABLE S10 Primers used for cloning and site saturationmutagenesis (SSM) of thrA. thrA_NcoI_F GCGCCATGGGAGTGTTGAAGTTCGGCGthrA_HindIII_R GCCAAGCTTTCAGACTCCTAACTTCCATGAGAGGG thrA_Y356NNK_FCGCAGAAACTGATGCTMNNTTCGGAAGATGATTGCG thrA_Y356NNK_RCGCAGAAACTGATGCTMNNTTCGGAAGATGATTGCG thrA_S357NNK_FCAATCATCTTCCGAATACNNKATCAGTTTCTGCGTTCC thrA_S357NNK_RGGAACGCAGAAACTGATMNNGTATTCGGAAGATGATTG thrA_S359NNK_FCCGAATACAGCATCNNKTTCTGCGTTCCAC thrA_S359NNK_RGTGGAACGCAGAAMNNGATGCTGTATTCGG

LIST OF REFERENCES CITED IN THE DESCRIPTION

-   Leuchtenberger W, Huthmacher K, Drauz K: Biotechnological production    of amino acids and derivatives: current status and prospects. Appl    Microbiol Biotechnol 2005, 69:1-8.-   Hagishita T, Yoshida T, Izumi Y, Mitsunaga T: Efficient L-serine    production from methanol and glycine by resting cells of    Methylobacterium sp. strain MN43. Biosci Biotechnol Biochem 1996,    60:1604-1607.-   Burgard A P, Maranas C D: Probing the performance limits of the    Escherichia coli metabolic network subject to gene additions or    deletions. Biotechnol Bioeng 2001, 74:364-375.-   Li Y, Chen G K, Tong X W, Zhang H T, Liu X G, Liu Y H, Lu F P:    Construction of Escherichia coli strains producing L-serine from    glucose. Biotechnol Lett 2012, 34:1525-1530.-   Peters-Wendisch P, Stolz M, Etterich H, Kennerknecht N, Sahm H,    Eggeling L: Metabolic engineering of Corynebacterium glutamicum for    L-serine production. Appl Environ Microbiol 2005, 71:7139-7144.-   Gu P, Yang F, Su T, U F, U Y, Qi Q: Construction of an L-serine    producing Escherichia coli via metabolic engineering. J Ind    Microbiol Biotechnol 2014, 41:1443-1450.-   Stolz M, Peters-Wendisch P, Etterich H, Gerharz T, Faurie R, Sahm H,    Fersterra H, Eggeling L: Reduced folate supply as a key to enhanced    L-serine production by Corynebacterium glutamicum. Appl Environ    Microbiol 2007, 73:750-755.-   Zhang X, Newman E: Deficiency in I-serine deaminase results in    abnormal growth and cell division of Escherichia coli K-12. Mol    Microbiol 2008, 69:870-881.-   Hama H, Sumita Y, Kakutani Y, Tsuda M, Tsuchiya T: Target of serine    inhibition in Escherichia coli. Biochem Biophys Res Commun 1990,    168:1211-1216.-   de Lorenzo V, Sekowska A, Danchin A: Chemical reactivity drives    spatiotemporal organisation of bacterial metabolism. FEMS Microbiol    Rev 2014.-   Chowdhury A, Zomorrodi A R, Maranas C D: k-OptForce: integrating    kinetics with flux balance analysis for strain design. PLoS Comput    Biol 2014, 10:e1003487.-   von Kamp A, Klamt S: Enumeration of smallest intervention strategies    in genome-scale metabolic networks. PLoS Comput Biol 2014,    10:e1003378.-   Qui Z and Goodman M F: The Escherichia coli polB locus is identical    to dinA, the structural gene for DNA polymerase II. Characterization    of Pol II purified from a polB mutant. J Biol Chem. 1997, 272(13):    8611-8617.-   Kwon D H, Peña J A, Osato M S, Fox J G, Graham D Y, Versalovic J:    Frameshift mutations in rdxA and metronidazole resistance in North    American Helicobacter pylori isolates. J Antimicrob Chemother 2000,    46(5): 793-796-   Yu D, Ellis H M, Lee E, Jenkins N A, Copeland N G, and Court D L: An    efficient recombination system for chromosome engineering in    Escherichia coli. Proc Natl Acad Sci USA 2000, 97: 5978-5983.-   Datsenko K A, Wanner B L: One-step inactivation of chromosomal genes    in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA    2000, 97:6640-6645.-   Sawitzke J A, Thomason L C, Bubunenko M, U X, Costantino N, Court D    L: Recombineering: using drug cassettes to knock out genes in vivo.    Methods Enzymol 2013, 533:79-102.-   Thomason L C, Costantino N, Court D L: E. coli genome manipulation    by P1 transduction. Curr Protoc Mol Biol 2007, Chapter 1:Unit 117.-   Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko K A,    Tomita M, Wanner B L, Mori H: Construction of Escherichia coli K-12    in-frame, single-gene knockout mutants: the Keio collection. Mol    Syst Biol 2006, 2:2006 0008.-   A I-Rabiee R, Zhang Y, Grant G A: The mechanism of velocity    modulated allosteric regulation in D-3-phosphoglycerate    dehydrogenase. Site-directed mutagenesis of effector binding site    residues. J Biol Chem 1996, 271:23235-23238.-   Kildegaard K R, Hallstrom B M, Blicher T H, Sonnenschein N, Jensen N    B, Sherstyk S, Harrison S J, Maury J, Herrgard M J, Juncker A S, et    al.: Evolution reveals a glutathione-dependent mechanism of    3-hydroxypropionic acid tolerance. Metab Eng 2014, 26C:57-66.-   Deatherage D E, Barrick J E: Identification of mutations in    laboratory-evolved microbes from next-generation sequencing data    using breseq. Methods Mol Biol 2014, 1151:165-188.-   Langmead B, Salzberg S L: Fast gapped-read alignment with Bowtie 2.    Nat Methods 2012, 9:357-359.-   Freddolino P L, Amini S, Tavazole S: Newly identified genetic    variations in common Escherichia coli MG1655 stock cultures. J    Bacteriol 2012, 194:303-306.-   Wang H H, Isaacs F J, Carr P A, Sun Z Z, Xu G, Forest C R, Church G    M: Programming cells by multiplex genome engineering and accelerated    evolution. Nature 2009, 460:894-898.-   Wang H H, Church G M: Multiplexed genome engineering and genotyping    methods applications for synthetic biology and metabolic    engineering. Methods Enzymol 2011, 498:409-426.-   Mertens N, Remaut E, Fiers W. 1995. Tight transcriptional control    mechanism ensures stable high-level expression from T7    promoter-based expression plasmids. Biotechnology (N Y) 13(2):175-9.-   St-Pierre F, Cui L, Priest D G, Endy D, Dodd I B, Shearwin    K E. 2013. One-step cloning and chromosomal integration of DNA. ACS    Synth Bol 2(9):537-41.

The invention claimed is:
 1. A bacterium belonging to theEnterobacteriaceae family, which has been modified to inactivate thegenes sdaA, sdaB, tdcG and glyA.
 2. The bacterium according to claim 1,wherein said bacterium has been further modified to overexpress a3-phosphoglycerate dehydrogenase, a phosphoserine phosphatase and aphosphoserine aminotransferase.
 3. The bacterium according to claim 1,wherein said bacterium is capable of growing in a minimal culture mediumcomprising L-serine at a concentration of at least about 6.25 g/L. 4.The bacterium according to claim 1, wherein said bacterium is capable ofgrowing in a minimal culture medium comprising L-serine at aconcentration of at least about 6.25 g/L at a growth rate of at least0.1 hr⁻¹ during exponential growth.
 5. The bacterium according to claim1, wherein said bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in one or more amino acidsubstitutions at a position selected from the group consisting of Y356,S357 and S359 in the amino acid sequence set forth in SEQ ID NO:
 11. 6.The bacterium according to claim 1, wherein said bacterium compriseswithin the thrA gene one or more nucleotide substitutions resulting inan amino acid substitution in the encoded polypeptide at position Y356,one or more nucleotide substitutions resulting in an amino acidsubstitution in the encoded polypeptide at position S357 and/or one ormore nucleotide substitutions resulting in an amino acid substitution inthe encoded polypeptide at position S359 in the amino acid sequence setforth in SEQ ID NO: 11; wherein the substitution at position Y356 isselected from the group consisting of Y356C, Y356T, Y356V, Y356S, Y356W,Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H,Y356R, and Y356L; the substitution at position S357 is selected from thegroup consisting of S357R, S357V, S357P, S357G, S357L, S357Y, S357A,S357N, S357F, S357H, S357K, S357I and S357M; and the substitution atposition S359 is selected from the group consisting of S359R, S359G,S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C, S359K, S359E andS359L.
 7. The bacterium according to claim 1, wherein said bacteriumexpress a polypeptide encoded by the thrA gene, wherein said polypeptidecomprises one or more amino acid substitutions in the amino acidsequence set forth in SEQ ID NO: 11 selected from the group consistingof Y356C, S357R and S359R.
 8. The bacterium according to claim 1,wherein said bacterium comprises within the thrA gene one or morenucleotide substitutions resulting in an amino acid substitution in theencoded polypeptide at position Y356, one or more nucleotidesubstitutions resulting in an amino acid substitution in the encodedpolypeptide at position S357 and/or one or more nucleotide substitutionsresulting in an amino acid substitution in the encoded polypeptide atposition S359 in the amino acid sequence set forth in SEQ ID NO: 11;wherein the substitution at position Y356 is selected from the groupconsisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N,Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R, and Y356L; thesubstitution at position S357 is selected from the group consisting ofS357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F, S357H,S357K, S357I and S357M; and the substitution at position S359 isselected from the group consisting of S359R, S359G, S359M, S359F, S359T,S359P, S359V, S359Q, S359A, S359C, S359K, S359E and S359L.
 9. Thebacterium according to claim 1, wherein said bacterium expresses apolypeptide having an amino acid sequence, which has at least about 90%sequence identity to the amino acid sequence set forth in SEQ ID NO: 11,which comprises an amino acid substitution at position Y356, S357 and/orS359, wherein the substitution at position Y356 is selected from thegroup consisting of Y356C, Y356T, Y356V, Y356S, Y356W, Y356Q, Y356G,Y356N, Y356D, Y356E, Y356F, Y356A, Y356I, Y356P, Y356H, Y356R and Y356L;the substitution at position S357 is selected from the group consistingof S357R, S357V, S357P, S357G, S357L, S357Y, S357A, S357N, S357F andS357H; and the substitution at position S359 is selected from the groupconsisting of S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q,S359A, S359C, S359K, S359E and S359L.
 10. The bacterium according toclaim 1, wherein said bacterium comprises an exogenous nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptide havingan amino acid sequence, which has at least about 90% sequence identityto the amino acid sequence set forth in SEQ ID NO: 11 and, whichcomprises an amino acid substitution at position Y356, S357 and/or S359.11. The bacterium according to claim 10, wherein the substitution atposition Y356 is selected from the group consisting of Y356C, Y356T,Y356V, Y356S, Y356W, Y356Q, Y356G, Y356N, Y356D, Y356E, Y356F, Y356A,Y356I, Y356P, Y356H, Y356R and Y356L; the substitution at position S357is selected from the group consisting of S357R, S357V, S357P, S357G,S357L, S357Y, S357A, S357N, S357F, S357H, S357K, S357I and S357M; andthe substitution at position S359 is selected from the group consistingof S359R, S359G, S359M, S359F, S359T, S359P, S359V, S359Q, S359A, S359C,S359K, S359E and S359L.
 12. The bacterium according to claim 1, whereinsaid bacterium has been further modified to overexpress the gene ydeD.13. The bacterium according to claim 1, wherein said bacterium comprisesone or more gene mutations selected from the group consisting of: one ormore nucleotide substitutions within the Irp gene resulting in the aminoacid substitution D143G in the amino acid sequence set forth in SEQ IDNO: 12, one or more nucleotide substitutions within the rho generesulting in the amino acid substitution R87L in the amino acid sequenceset forth in SEQ ID NO: 13, one or more nucleotide substitutions withinthe eno gene resulting in the amino acid substitution V164L in the aminoacid sequence set forth in SEQ ID NO: 14, one or more nucleotidesubstitutions within the argP gene resulting in the amino acidsubstitution Q132K in the amino acid sequence set forth in SEQ ID NO:15, one or more nucleotide substitutions within the tufA gene resultingin the amino acid substitution G19V in the amino acid sequence set forthin SEQ ID NO: 16, one or more nucleotide substitutions within the cycAgene resulting in the amino acid substitution 1220V in the amino acidsequence set forth in SEQ ID NO: 17, one or more nucleotidesubstitutions within the rpe gene resulting in the amino acidsubstitution I202T in the amino acid sequence set forth in SEQ ID NO:18, one or more nucleotide substitutions within the yojl gene resultingin the amino acid substitution D334H in the amino acid sequence setforth in SEQ ID NO: 19, one or more nucleotide substitutions within thehyaF gene resulting in the amino acid substitution V120G in the aminoacid sequence set forth in SEQ ID NO: 20, one or more nucleotidesubstitutions within the pykF gene resulting in the amino acidsubstitution E250* in the amino acid sequence set forth in SEQ ID NO:21, where * designates a stop codon, one or more nucleotidesubstitutions within the malT gene resulting in the amino acidsubstitution Q420* in the amino acid sequence set forth in SEQ ID NO:23, where * designates a stop codon, one or more nucleotidesubstitutions within the rpoB gene resulting in the amino acidsubstitution P520L in the amino acid sequence set forth in SEQ ID NO:25, one or more nucleotide substitutions within the fumB gene resultingin the amino acid substitution T218P in the amino acid sequence setforth in SEQ ID NO: 26, one or more nucleotide substitutions within thegshA gene resulting in the amino acid substitution A178V in the aminoacid sequence set forth in SEQ ID NO: 27, and one or more nucleotidesubstitutions within the lamB gene resulting in the amino acidsubstitution Q112* in the amino acid sequence set forth in SEQ ID NO:28, where * designates a stop codon.
 14. The bacterium according toclaim 1, wherein said bacterium belongs to the genus Escherichia. 15.The bacterium according to claim 1, wherein said bacterium isEscherichia coli.
 16. A method for producing L-serine or a L-serinederivative, the method comprising cultivating a bacterium according toclaim 1 in a culture medium.
 17. The method according to claim 16,wherein the L-serine derivative is selected from the group consisting ofL-cysteine, L-methionine, L-glycine, O-acetylserine, L-tryptophan,thiamine, ethanolamine and ethylene glycol.